Human Brain Microvascular Cells Research Articles

Inhibiting Caveolin-1-Related Akt/mTOR Signaling Pathway Protects Against N-methyl-D-Aspartate Receptor Activation-Mediated Dysfunction of Blood–Brain Barrier in vitro

Abstract Background The aim of this study was to further explore the role of caveolin-1 (Cav-1) related Akt/mTOR signaling pathway in blood brain barrier (BBB) dysfunction caused by NMDAR activation. Methods The cell localization of NMDAR GluN1 subunit and Cav-1 was observed on human brain microvascular HBEC-5i cells after immunofluorescence double staining. The transendothelial resistance (TEER) of BBB in vitro was measured by Millicell-ERS cell resistance meter. Sodium fluorescein (SF) was used to measure the permeability of BBB in vitro. A stable Cav-1-silenced HBEC-5i cell line was established by infecting the cells with a lentivirus encoding Cav-1 shRNA. The changes of the protein and mRNA of MMP9 and Occludin induced by NMDA were detected by Western blot (WB) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. The phosphorylated proteins of Cav-1, Akt, and mTOR were detected by WB. Results NMDAR GluN1 was expressed in the cytoplasm and part of the cell membrane of the HBEC-5i cell line. NMDAR activation decreased TEER and increased the SF of BBB in vitro. HBEC-5i cells incubated with NMDA enhanced the phosphorylation of Cav-1, Akt, and mTOR, also promoting the expression of MMP9 along with the degradation of Occludin. These effects could be reversed by pretreatment with NMDAR antagonist (MK801) or Cav-1 antagonist (Daidzein), or Akt antagonist (LY294002), respectively. Further silencing Cav-1 with LV-Cav-1-RNAi also played a similar protective effect. Conclusion Caveolin-1 (Cav-1) related Akt/mTOR signaling probably contributes to BBB dysfunction by activating NMDAR on human brain microvascular cells.

Association of Apolipoprotein E4-related Microvascular Disease in the Alzheimer’s Disease Hippocampal CA1 Stratum Radiatum

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer’s disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-β (Aβ) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.

Neuregulin-1 attenuates hemolysis- and ischemia induced-cerebrovascular inflammation associated with sickle cell disease

ObjectivesIndividuals with sickle cell disease (SCD) are at severely heightened risk for cerebrovascular injury and acute cerebrovascular events, including ischemic and hemorrhagic stroke, potentially leading to impaired development and life-long physical and cognitive disabilities. Cerebrovascular injury specific to SCD includes inflammation caused by underlying conditions of chronic hemolysis and reduced cerebrovascular perfusion. The objectives of this study were to investigate whether expression of neuregulin-1β (NRG-1), an endogenous neuroprotective polypeptide, is increased in SCD or experimental conditions mimicking the hemolysis and ischemic conditions of SCD, and to determine if treatment with exogenous NRG-1 reduces markers of cerebrovascular inflammation. Materials and methodsPlasma and brain-specific NRG-1 levels were measured in transgenic SCD mice. Endogenous NRG-1 levels and response to experimental conditions of excess heme and ischemia were measured in cultured human brain microvascular cells and astrocytes. Pre-treatment with NRG-1 was used to determine NRG-1’s ability to ameliorate resultant cerebrovascular inflammation. ResultsPlasma and brain-specific NRG-1 were elevated in transgenic SCD mice compared to healthy controls. Neuregulin-1 expression was significantly increased in cultured human microvascular cells and astrocytes exposed to excess heme and ischemia. Pre-treatment with NRG-1 reduced inflammatory chemokine (CXCL-1 and CXCL-10) and adhesion molecule (ICAM-1 and VCAM-1) expression and increased pro-angiogenic factors (VEGF-A) in microvascular cells and astrocytes exposed to excess heme and ischemia. ConclusionsElevated NRG-1 in SCD is likely a protective endogenous response to ongoing cerebrovascular insults caused by chronic hemolysis and reduced cerebrovascular perfusion. Administration of NRG-1 to reduce cerebrovascular inflammation may be therapeutically beneficial in SCD and warrants continued investigation.

Blast-induced injury responsive relative gene expression of traumatic brain injury biomarkers in human brain microvascular endothelial cells

Disruption of the blood–brain barrier (BBB) is a critical component of traumatic brain injury (TBI) progression. However, further research into the mechanism of BBB disruption and its specific role in TBI pathophysiology is necessary. To help make progress in elucidating TBI affected BBB pathophysiology, we report herein relative gene expression of eleven TBI biomarkers and other factors of neuronal function in human brain microvascular cells (HBMVEC), one of the main cell types in the BBB. Our in-vitro blast TBI model employs a custom acoustic shock tube to deliver injuries of varying intensities to HBMVECs in culture. Each of the investigated genes exhibit a significant change in expression as a response to TBI, which is dependent on both the injury intensity and time following the injury. This data suggests that cell signaling of HBMVECs could be essential to understanding the interaction of the BBB and TBI pathophysiology, warranting future investigation.

β-Catenin Restricts Zika Virus Internalization by Downregulating Axl.

The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, β-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited β-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that β-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for β-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and β-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that β-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates β-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits β-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

Valsartan alleviates the blood–brain barrier dysfunction in db/db diabetic mice

ABSTRACT Type 2 diabetes (T2D)-related neurological complication is the risk factor for neurodegenerative disorders. The pathological changes from T2D-caused blood–brain barrier (BBB) dysfunction plays a critical role in developing neurodegeneration. The hyper-activation of the Angiotensin II type 1 receptor (AT1R) in the brain is associated with neurovascular impairment. The AT1R antagonist Valsartan is commonly prescribed to control high blood pressure, heart failure, and diabetic kidney diseases. In this study, we investigated the beneficial effects of Valsartan in db/db diabetic mice and isolated brain endothelial cells. We showed that 2 weeks of Valsartan administration (30 mg/Kg body weight) mitigated the increased permeability of the brain-blood barrier and the reduction of gap junction proteins VE-Cadherin and Claudin 2. In human brain microvascular cells (HBMVECs), we found that Valsartan treatment ameliorated high glucose-induced hyperpermeability by measuring Dextran uptake and transendothelial electrical resistance (TEER). Furthermore, Valsartan treatment recovered high glucose-repressed endothelial VE-Cadherin and Claudin 2 expression. Moreover, Valsartan significantly suppressed the expressions of pro-inflammatory cytokines such as macrophage chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) against high glucose. Mechanistically, Valsartan ameliorated high glucose-repressed endothelial cAMP-responsive element-binding protein (CREB) signaling activation. The blockage of CREB activation by PKA inhibitor H89 abolished the action of Valsartan, suggesting its dependence on CREB signaling. In conclusion, Valsartan shows a neuroprotective effect in diabetic mice by ameliorating BBB dysfunction. These effects of Valsartan require cellular CREB signaling in brain endothelial cells.

Flow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with an increased rate of cerebrovascular events including ischemic stroke and intracerebral hemorrhage. The mechanisms underlying cerebral endothelial susceptibility and response to SARS-CoV-2 are unknown yet critical to understanding the association of SARS-CoV-2 infection with cerebrovascular events. Endothelial cells were isolated from human brain and analyzed by RNA sequencing. Human umbilical vein and human brain microvascular cells were used in both monolayer culture and endothelialized within a 3-dimensional printed vascular model of the middle cerebral artery. Gene expression levels were measured by quantitative polymerase chain reaction and direct RNA hybridization. Recombinant SARS-CoV-2 S protein and S protein-containing liposomes were used to measure endothelial binding by immunocytochemistry. ACE2 (angiotensin-converting enzyme-2) mRNA levels were low in human brain and monolayer endothelial cell culture. Within the 3-dimensional printed vascular model, ACE2 gene expression and protein levels were progressively increased by vessel size and flow rates. SARS-CoV-2 S protein-containing liposomes were detected in human umbilical vein endothelial cells and human brain microvascular endothelial cells in 3-dimensional middle cerebral artery models but not in monolayer culture consistent with flow dependency of ACE2 expression. Binding of SARS-CoV-2 S protein triggered 83 unique genes in human brain endothelial cells including upregulation of complement component C3. Brain endothelial cells are susceptible to direct SARS-CoV-2 infection through flow-dependent expression of ACE2. Viral S protein binding triggers a unique gene expression profile in brain endothelia that may explain the association of SARS-CoV-2 infection with cerebrovascular events.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Function of gene 0267 of Streptococcus suis type 2 98HAH33

Objective To study the function of gene 0267 of Streptococcus suis type 2 98HAH33. Methods Growth rates of the wild type strain Streptococcus suis type 2 98HAH33, the mutant stain Δ0267 and the complemented strain CΔ0267 at different stages were compared. Bacterial adhesion, whole blood killing and macrophage phagocytosis assays were used to compare the adhesion and anti-phagocytosis of these strains to host cells. A piglet model was used to evaluate their differences in virulence. Results The growth rates of the wild type strain, the mutant strain and the complemented strain were the same. The adhesion rates of the wild type strain and the complemented strain to A549 cells, Hep2 cells and human brain microvascular endothelial cells (HBMEC) were 2.59, 4.87 and 3.08 times, and 2.65, 4.65 and 2.86 times higher than those of the mutant strain, respectively. The survival rates at 1 h, 2 h and 3 h of the wild type strain, the mutant strain and the complemented strain in whole blood were 36.91%, 28.12% and 41.61%, 58.44%, 44.06% and 58.26%, and 93.02%, 70.08% and 95.85%, respectively. Results of the phagocytosis assay showed that the bacterial loads of the wild type strain, the mutant strain and the complemented strain in murine RAW264.7 macrophages were 2 767, 5 322 and 2 567, respectively. The competitive infection experiment using piglets showed that gene 0267 was associated with the virulence of Streptococcus suis type 2 98HAH33. Conclusions Streptococcus suis type 2 98HAH33 gene 0267 is associated with bacterial adhesion and anti-phagocytosis, suggesting that it is a new virulence factor. Key words: Streptococcus suis type 2; Bacterial adhesion; Whole blood killing; Phagocytosis; Virulence factor

The biological effects of magnetic fields on microvascular endothelial cells in the human brain

Objective To observe the biological effects of magnetic fields of different intensities on microvascular endothelial cells in the human brain (HBMECs). Methods HBMECs were cultured in vitro under normal conditions and randomly divided into a control group and several magnetic induction groups——26 mT, 62.5 mT, 110.7 mT and 215.6 mT at the center pole. Any changes to the cytomembranes were observed 72 h after planking using the lactate dehydrogenase (LDH) method. Superoxide dismutase and malondialdehyde methods were used to detect cellular oxidation due to the magnetic field. An inverted microscope was used to observe any changes in cell morphology, and flow cytometry was employed to detect cell apoptosis. Results Compared with the control group, the LDH value of the 215.6 mT group was significantly higher, but there were no significant differences in oxidative damage, apoptosis or morphology observed. Moreover, there were no significant differences between the controls and the 26 mT, 62.5 mT and 110.7 mT groups in any of the above measurements. Conclusion Magnetic fields of different intensity have different biological effects on HBMECs. A 215.6 mT magnetic field influences their cell membranes but causes no oxidative damage, cell apoptosis or morphological changes. These observations and the mechanism need further exploration. Key words: Magnetic induction; Human brain microvascular endothelial cells; Oxidative damage; Cell apoptosis; Cell morphology

Aloe-emodin triggers ROS and Ca 2+ production and decreases the levels of mitochondrial membrane potential of human brain capillary endothelial cells

<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>

The effects of Celecoxib on human brain microvascular endothelial cells release 6-keto-PGF1α and TXB2 and apoptosis after radiation

Objective To investigate the effect of Celecoxib on human brain microvascular endothelial cells release6-keto-PGF1α, TXB2 and apotosis after irradiation. Methods The logarithmic growth phase cells were divided into control groups (Con), simple irradiation (IR) groups and combination groups (IR+ C). CCK-8 and clone formation experiment were used to evaluate the effects of radiosensitivity and toxicity of celecoxib. The results were observed atthe time point of 6 h, 12 h, 24 h, 48 h after irradiation. ELISA was used to test the contents of 6-keto-PGF1α and TXB2, which metabolized by PGI2 and TXA2 from culture medium after irradiation at different time points in different groups. TXB2/6-keto-PGF1αratios were calculated. Annexin V-FITC/PI double staining method was used to measure the apoptosis rates at different time points in different groups. Western blot was used to measure the protein expression. Paired t test difference. Results Compared with simple irradiation group, there were no significant radiosensitivity (SER=0.96) in combination groups incubated with30 μmol/L of celecoxib. Compared with the control group, the ratio of TXB2/6-keto-PGF1αincreased at each time point in IR and IR+ C (P<0.05), and the apoptosis rates increased (P<0.05). Cox-2, P-JNK and Cleaved caspase-3 increased. Compared with IR, the ratio of TXB2/6-keto-PGF1αdecreased at each time point in IR+ C (P<0.05), and the apoptosis rates decreased (t=3.34~6.38, P< 0.05). The protein expression of Cox-2, P-JNK and Cleaved caspase-3 decreased. Conclusions Celecoxib may help to protect HBMECs from releasing TXA2 and decreasing the ratio of TXB2/6-keto-PGF1α, and inhibitting apoptosis after irradiation. The mechanisms of apoptosis inhibition may be related to the inhibition of Cox-2 and P-JNK, caspase-3 Cleaved proteinexpressions. Key words: Human brain microvascular endothelial cells; Cox-2; Celecoxib; Irradiation; Prostacyclin; Thromboxane; Apoptosis

Sphingosine‐1‐phosphate and 8‐pCPT‐2′‐O‐Me‐cAMP Can Restore Alcohol‐induced Junctional Disorganization and Barrier Dysfunction of Human Brain Microvascular Endothelial Cells

The blood‐brain‐barrier (BBB) is a highly selective endothelial barrier that protects the brain from blood‐born cytotoxins and ensures proper homeostasis of the central nervous system. Alcohol has previously been shown to compromise the BBB, however the mechanisms involved are incompletely understood. We hypothesized that alcohol increases BBB permeability by activating the p38 MAP kinase, disrupting paracellular junction proteins, such as VE‐Cadherin and Claudin‐5, and that microvascular barrier stabilizers such as sphingosine‐1‐phosphate (S1P) and 8‐pCPT‐2′‐O‐Me‐cAMP (8‐CPT) can resolve alcohol‐induced BBB dysfunction. Cultured primary human brain microvascular endothelial cell (HBMEC) monolayers were used to model BBB function so that endothelial‐specific mechanisms could be studied. Trans‐endothelial electrical resistance (TER) was used as an index of barrier function. Localization of VE‐cadherin and Claudin‐5 was determined by immunofluorescence microscopy. S1P (1 μM) or 8‐CPT (100 μM) were applied following treatment with alcohol to test their efficacies to rescue barrier function. The results show that treatment of HBMEC with alcohol (50–100 mM) causes a biphasic hyperpermeability response, characterized by an initial transient decrease in TER (10 min) that recovers, followed by a prolonged decrease in TER that lasts several hours. Surprisingly, we observed that pretreatment with the selective p38 MAPK inhibitor SB208530 (20 μM) does not block the alcohol‐induced decrease in HBMEC monolayer TER. Predictably, treatment of HBMEC monolayers with either S1P or 8‐CPT significantly enhances barrier function. However, pretreatment with S1P or 8‐CPT does not prevent the disruption caused by 75 mM alcohol, and S1P pretreatment can even exacerbate the alcohol‐induced decrease in TER. In contrast, application of S1P or 8‐CPT, after alcohol has already evoked a decrease in TER, causes a hastened recovery to normal HBMEC monolayer barrier function. Our Immunocytochemistry studies demonstrate colocalization of VE‐Cadherin and Claudin‐5 at intercellular junctions, and alcohol disrupts this organization. Importantly, treatment with S1P (after alcohol) can restore junctional integrity. Taken together, our results show that alcohol causes a biphasic decrease in HBMEC monolayer barrier function that does not require p38 MAP kinase activation, but does involve reorganization of junctional protein complexes. In addition, S1P and 8‐CPT can enhance barrier function and may be useful for restoring BBB function in the presence of alcohol intoxication.Support or Funding InformationSupported by a grant from the USF MCOM Office of Research.

Attachment of Treponema pallidum to human brain microvascular endothelial cells in vitro: an experimental study

Objective To observe the attachment of Treponema pallidum to human brain microvascular endothelial cells (HBMECs) in vitro. Methods Some primary cultured HBMECs were inoculated into in 24-well plates to be cocultured with the suspension of T. pallidum at a concentration of 1.6 × 107 treponemes/ml. After 0.5, 2 and 4 hours of co-culture, scanning electron microscopy was conducted to observe the attachment of T. pallidum to HBMECs. Some HBMECs were cocultured with the presence of T. pallidum suspensions at different concentrations (4 × 106, 8 × 106, 1.6 × 107 treponemes/ml) for 2, 4, 6 and 16 hours, then, dark-field microscopy was performed to count the number of treponemes that attached to single HBMECs. Statistical analysis was carried out by using repeated-measures analysis of variance. Results As scanning electron microscopy showed, treponemes gathered at some regions on the surface of HBMECs when they attached to HBMECs. In addition, T. pallidum partly merged with the membrane of HBMECs at the site of attachment. After co-culture with T. pallidum suspensions, the number of treponemes that attached to single HBMECs was significantly different among different time points (F= 387.72, P< 0.001) and among different concentrations of T. pallidum suspensions (F= 593.23, P< 0.001) , with an interaction effect between the concentration of T. pallidum suspensions and incubation period (F= 98.74, P< 0.001). Concretely speaking, the number of treponemes that attached to single HBMECs increased over time until 6 hours after the start of coculture, then showed a decreasing trend, and reached the nadir value at 16 hours. Conclusion T. pallidum can adhere to cultured HBMECs in vitro, likely by the merger of its end with the membrane of HBMECs at some regions. Key words: Treponema pallidum; Endothelial cells; Microvessels; Human brain microvascular endothelial cells

Abstract W P407: Shear Stress and Co-culture with Astrocytes Determine Brain Microvascular Endothelial Cell Phenotype

Introduction: Dementia has classically been identified to be of either vascular or neural origin. These domains overlap and are complementary, thus we consider dementia a disease of the single cerebrovascular unit. Our objective was to generate a modular platform for co-culture of brain microvascular endothelial cells and astrocytes that also incorporates mechanical stresses, and to then use this model of the cerebrovascular unit microenvironment to study the unit in vitro . Hypothesis: We assessed the hypothesis that endothelial health and blood-brain barrier integrity are modulated by shear stress and co-culture with astrocytes. Methods: We lithographed polydimethylsiloxane substrate on Teflon negative molds with subjacent rectangular channels of 0.45 and 2 mm depth for seeding of human brain microvascular cells and astrocytes, respectively, separated by a polytetrafluoroethylene (0.45 μm pore size) membrane under no flow or physiologic flow (6.2 dynes/cm 2 ) for one week. Immunocytochemical staining for glial fibrillary acidic protein and CD31 was simultaneously visualized by confocal microscopy. Cells from each channel were detached via trypsinization, and expression of transport proteins P-glycoprotein (P-gp) and glucose transporter-1 (GLUT-1), in addition to junction proteins zona occludens-1 (ZO-1) and CD31, was measured by Western Blot. Results: We stably co-cultured brain microvascular endothelial cells and astrocytes with no chamber leakage or mixing. CD31 staining revealed endothelial cell alignment to direction of flow. Expression of ZO-1 by endothelial cells increased in presence of flow and co-culture independently, by 1.6-fold in combined conditions relative to static monoculture (p&lt;0.05). For P-gp, the increase in combined conditions was 5.5-fold (p&lt;0.05). GLUT-1 and CD31 levels did not change significantly with co-culture or flow. Conclusion: Cell biology devoid of microenvironmental cues provides limited insight, especially when considering whole tissues, on the impact of disease. A co-culture system that introduces multiple cells, flow, controlled stress and independent visualization and sampling of each cell domain adds deeper understanding and greater value to in vitro biological models and tissue biology.

Effects of permanent magnetic fields on superoxide dismutase and malondialdehyde in human microvascular endothelial brain cells under normal and hypoxic conditions

Objective To observe the effects of permanent magnetic fields with different magnetic inductions on oxidative damage to human brain microvascular endothelial cells (HBMECs) under normal and hypoxic conditions. Methods HBMECs were cultured in vitro under normal and hypoxic conditions,then divided into a control group and groups receiving magnetic induction at 8.1,16.5,20.3,26.0,27.3,62.5,110.7 and 215.6 mT.Changes in superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in each group were measured 72 h after exposure to the magnetic fields. Results Under normal conditions the SOD activities of the magnetic groups were not significantly different from that of the control group. Only the MDA content of the 215.6 mT group was statistically different (slightly higher) than that of the control group (P ＜ 0.05 ).Under hypoxic conditions,there was again no statistically significant difference in SOD activity,but the MDA content of the 110.7 mT group was significantly lower. Conclusions Under normal conditions,a 215.6 mT permanent magnetic field has an effect on MDA in HBMECs; under hypoxic conditions a 110.7 mT permanent magnetic field has such an effect. Key words: Permanent magnetic fields; Microvascular endothelial brain cells; Superoxide dismutase; Malondialdehyde

Efficacy of genetically engineered oncolytic virus on glioma stem cells in vitro

Objective Exploring the effect of endostatin and angiostatin(Endo-Angio) fusion genemodified herpes simplex virus ( VAE ) on glioma stem cells ( GSCs ) in vitro. Method Surgical specimens of human high grade glioma ( WHO Ⅲ, Ⅵ) were collected, and GSCs were isolated under the culture conditionsoriginally designed for selective expansion of neural stem cells. In order to identify GSCs,the number of CD133 positive cells in GSCs were detected by flow cytometry; self - renewal capacity and the expression of Nestin, GFAP, NF, and MAG of GSCs were detected by monoclonal forming and immunofluorescence assay respectively. After that, GSCs viability was detected by MTS assay. Expression of Endo - Angio fusion gene at mRNA and protein level was detected by RT - PCR and Western blot. At last,the efficacy of fusion protein to human brain microvascular endothelial cells (HBMEC) and the biological properties of GSCs were detect after infected by VAE. Results ( 1 )4 GSCs isolated from 20 surgical specimens could suspend growth and had the ability of self-renewal and multipotential differentiation,and could express neural stem cells marker,CD133 and Nestin. (2)VAE could infect GSCs and significantly inhibit the viability of GSCs( P ＜0.05). (3) Significant expression of Endo-Angio fusion gene was observed and it could inhibit HBMEC proliferation( P ＜0.05). (4) Residual viable cells lost the ability of self - renewal and adherent differentiation. Conclusions In vitro, VAE can inhibit the activity of GSCs and the expression of exogenous Endo - Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novd virus -gene therapy strategy for glioma. Key words: Glioma; Neoplastic stem cells; Endostatins; Angiogenesis inhibitors; Virus - gene therapy

Impaired Orthotopic Glioma Growth and Vascularization in Transgenic Mouse Models of Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia among the aging population and is characterized pathologically by the progressive intracerebral accumulation of beta-amyloid (Abeta) peptides and neurofibrillary tangles. The level of proangiogenic growth factors and inflammatory mediators with proangiogenic activity is known to be elevated in AD brains which has led to the supposition that the cerebrovasculature of AD patients is in a proangiogenic state. However, angiogenesis depends on the balance between proangiogenic and antiangiogenic factors and the brains of AD patients also show an accumulation of endostatin and Abeta peptides which have been shown to be antiangiogenic. To determine whether angiogenesis is compromised in the brains of two transgenic mouse models of AD overproducing Abeta peptides (Tg APPsw and Tg PS1/APPsw mice), we assessed the growth and vascularization of orthotopically implanted murine gliomas since they require a high degree of angiogenesis to sustain their growth. Our data reveal that intracranial tumor growth and angiogenesis is significantly reduced in Tg APPsw and Tg PS1/APPsw mice compared with their wild-type littermates. In addition, we show that Abeta inhibits the angiogenesis stimulated by glioma cells when cocultured with human brain microvascular cells on a Matrigel layer. Altogether our data suggest that the brain of transgenic mouse models of AD does not constitute a favorable environment to support neoangiogenesis and may explain why vascular insults synergistically precipitate the cognitive presentation of AD.

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells.

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.