Rat Brain Microvascular Endothelial Cells Research Articles

Curcumin attenuates ischemia-like injury induced IL-1β elevation in brain microvascular endothelial cells via inhibiting MAPK pathways and nuclear factor-κB activation

Inflammatory reactions play a key role in the cerebral injury after stroke or other ischemic brain diseases. Curcumin, which is extracted from herb turmeric, has been reported to have anti-inflammatory effects. The present study was aimed to investigate the anti-inflammatory effects of curcumin on oxygen-glucose deprivation (OGD) injured brain microvascular endothelial cells (BMECs). Rat BMECs were used and the results showed that OGD induced a significant elevation of the leakage of lactate dehydrogenase and the secretion of the proinflammation cytokine, IL-1β. Activation of p38, JNK MAPKs, and NF-κB in BMECs was also observed after OGD. The treatment of curcumin (20 μM) inhibited the increased production of IL-1β both at the protein and mRNA levels. The increased phosphorylation of p38 and JNK induced by OGD was decreased under the treatment of curcumin, whereas the p38 inhibitor, SB203580, significantly inhibited OGD-induced IL-1β production, but the JNK inhibitor, SP600125, failed to do so. These results suggest that the inhibition of IL-1β by curcumin may dependent on the p38 signaling pathway. The OGD-induced IL-1β production was also inhibited by the NF-κB inhibitor, and curcumin suppressed OGD-induced NF-κB activation. Furthermore, the NF-κB activation was attenuated by the SB203580, indicating that NF-κB activation was dependent on p38 signaling pathway. The present study suggests that curcumin displays an anti-inflammatory effect on OGD-injured BMECs via down-regulating of MAPK and NF-κB signaling pathways and might have therapeutic potential for the ischemic brain diseases.

Bradykinin regulates the expression of claudin‐5 in brain microvascular endothelial cells via calcium‐induced calcium release

To investigate the mechanism underlying the regulation of claudin-5, a tight junction protein that participates primarily in the constitution of the blood-brain barrier by bradykinin (BK), we established a primary culture of rat brain microvascular endothelial cells (BMECs). BMECs were treated with 10(-5) M BK, and changes in the intracellular Ca(2+) levels were measured by using the sensitive fluorescent dye fluo-3; the expression and distribution of claudin-5 were investigated by immunocytochemistry and Western blot analyses. We did not detect any expression of bradykinin B2 receptors in the BMECs or freshly isolated rat brain microvessels. We found that 10(-5) M BK triggered Ca(2+) transients in BMECs, and further investigations revealed that inositol 1,4,5-trisphosphate receptors (IP3 Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum (ER) were responsible for the Ca(2+) fluctuation. Consequently, these intracellular Ca(2+) changes that occur in response to BK application were identified as Ca(2+) -induced Ca(2+) release (CICR). Immunocytochemistry and Western blot results demonstrated that 10(-5) M BK could cause the internalization and a decrease in the expression of claudin-5; agonists of IP3 Rs and RyRs, such as IP3 and caffeine, enhanced the BK-induced downregulation of claudin-5, whereas antagonists of IP3 Rs and RyRs, such as 2-APB and ryanodine, abrogated BK's effect on claudin-5. In conclusion, the BK-induced CICR in primary culture BMECs might be the mechanism by which BK modulates claudin-5.

Regulation of monocarboxylic acid transporter-1 by cAMP dependent vesicular trafficking in brain microvascular endothelial cells.

In this study, a detailed characterization of Monocarboxylic Acid Transporter-1 (Mct1) in cytoplasmic vesicles of cultured rat brain microvascular endothelial cells shows them to be a diverse population of endosomes intrinsic to the regulation of the transporter by a brief 25 to 30 minute exposure to the membrane permeant cAMP analog, 8Br-cAMP. The vesicles are heterogeneous in size, mobility, internal pH, and co-localize with discreet markers of particular types of endosomes including early endosomes, clathrin coated vesicles, caveolar vesicles, trans-golgi, and lysosomes. The vesicular localization of Mct1 was not dependent on its N or C termini, however, the size and pH of Mct1 vesicles was increased by deletion of either terminus demonstrating a role for the termini in vesicular trafficking of Mct1. Using a novel BCECF-AM based assay developed in this study, 8Br-cAMP was shown to decrease the pH of Mct1 vesicles after 25 minutes. This result and method were confirmed in experiments with a ratiometric pH-sensitive EGFP-mCherry dual tagged Mct1 construct. Overall, the results indicate that cAMP signaling reduces the functionality of Mct1 in cerebrovascular endothelial cells by facilitating its entry into a highly dynamic vesicular trafficking pathway that appears to lead to the transporter's trafficking to autophagosomes and lysosomes.

Hepatocyte growth factor enhances the barrier function in primary cultures of rat brain microvascular endothelial cells

The effects of hepatocyte growth factor (HGF) on barrier functions were investigated by a blood–brain barrier (BBB) in vitro model comprising a primary culture of rat brain capillary endothelial cells (RBEC). In order to examine the response of the peripheral endothelial cells to HGF, human umbilical vascular endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) were also treated with HGF. HGF decreased the permeability of RBEC to sodium fluorescein and Evans blue albumin, and dose-dependently increased transendothelial electrical resistance (TEER) in RBEC. HGF altered the immunochemical staining pattern of F-actin bands and made ZO-1 staining more distinct on the linear cell borders in RBEC. In contrast, HGF increased sodium fluorescein and Evans blue albumin permeability in HMVEC and HUVEC, and decreased TEER in HMVEC. In HMVEC, HGF reduced cortical actin bands and increased stress fiber density, and increased the zipper-like appearance of ZO-1 staining. Western blot analysis showed that HGF significantly increased the amount of ZO-1 and VE-cadherin. HGF seems to act on the BBB to strengthen BBB integrity. These findings indicated that cytoskeletal rearrangement and cell–cell adhesion, such as through VE-cadherin and ZO-1, are candidate mechanisms for the influence of HGF on the BBB. The possibility that HGF has therapeutic significance in protecting the BBB from damage needs to be considered.

Increased Susceptibility to Amyloid-β Toxicity in Rat Brain Microvascular Endothelial Cells under Hyperglycemic Conditions

We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-β peptide (Aβ) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Leprdb/Leprdb) and heterozygous (Dock7m/Leprdb) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aβ1-40. Some experiments were also performed with two mitochondrial superoxide (O2•-) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔΨm) by confocal microscopy. Mitochondrial O2•- and hydrogen peroxide (H2O2) production was assessed by fluorescence microscopy and H2O2 production was confirmed by microplate reader. Hyperglycemia or Aβ1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aβ1-40 reduced cell viability and ΔΨm, and enhanced mitochondrial O2•- and H2O2 production. MitoTempo and PEG-SOD prevented Aβ1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aβ1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aβ toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.

Low-dose Endothelial Monocyte-activating Polypeptide-II Increases Permeability of Blood–tumor Barrier by Caveolae-mediated Transcellular Pathway

Low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase blood-tumor barrier (BTB) permeability via the paracellular pathway. The role of the transcellular pathway in this process is unclear. This study was conducted to evaluate the potential involvement of the transcellular pathway in EMAP-II-induced opening of the BTB and to identify the associated mechanisms. Evans blue extravasation test was used to measure changes in BTB permeability after EMAP-II (80ng/kg) administration in a rat model of C6 glioma. Changes in the quantity of pinocytotic vesicles in rat brain microvascular endothelial cells (BMECs) were observed using transmission electron microscopy. Reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry assays were performed to detect the expression of the caveolar structural proteins, caveolin-1 and caveolin-2, in BMECs. Alterations in the expression of phospho (p)-Src, p-caveolin-1, and p-caveolin-2 and the activity of RhoA also were measured. Effects of tyrosine kinase inhibition on EMAP-II-induced RhoA/Rho kinase activations and tyrosine kinase, RhoA, or Rho kinase inhibition on EMAP-II-induced caveolin-1 and caveolin-2 phosphorylation were determined by inhibition studies. One hour after EMAP-II administration, the quantity of pinocytotic vesicles in BMECs increased markedly, consistent with changes in BTB permeability. The expression levels of caveolin-1, caveolin-2, p-caveolin-1, and p-caveolin-2 in BMECs also were significantly increased at 1h. The peak expression level of p-Src and the peak activity of RhoA occurred at 0.25 and 0.5h, respectively. Inhibition of tyrosine kinase significantly diminished the activities of RhoA and Rho kinase induced by EMAP-II. In addition, EMAP-II-induced phosphorylation of caveolin-1 and caveolin-2 was completely blocked by inhibition of tyrosine kinase, RhoA, or Rho kinase. We suggest that low-dose EMAP-II can induce BTB hyperpermeability via the transcellular pathway, which is associated with phosphorylation and upregulation of caveolin-1 and caveolin-2 and involves the tyrosine kinase/RhoA/Rho kinase signaling pathway.

Ion transporters in brain endothelial cells that contribute to formation of brain interstitial fluid

Ions and water transported across the endothelium lining the blood–brain barrier contribute to the fluid secreted into the brain and are important in maintaining appropriate volume and ionic composition of brain interstitial fluid. Changes in this secretion process may occur after stroke. The present study identifies at transcript and protein level ion transporters involved in the movement of key ions and examines how levels of certain of these alter following oxidative stress. Immunohistochemistry provides evidence for Cl−/HCO3 − exchanger, AE2, and Na+, HCO3 − cotransporters, NBCe1 and NBCn1, on brain microvessels. mRNA analysis by RT-PCR reveals expression of these transporters in cultured rat brain microvascular endothelial cells (both primary and immortalized GPNT cells) and also Na+/H+ exchangers, NHE1 (primary and immortalized) and NHE2 (primary cells only). Knock-down using siRNA in immortalized GPNT cells identifies AE2 as responsible for much of the Cl−/HCO3 − exchange following extracellular chloride removal and NHE1 as the transporter that accounts for most of the Na+/H+ exchange following intracellular acidification. Transcript levels of both AE2 and NHE1 are increased following hypoxia/reoxygenation. Further work is now required to determine the localization of the bicarbonate transporters to luminal or abluminal membranes of the endothelial cells as well as to identify and localize additional transport mechanisms that must exist for K+ and Cl−.

A simple method for isolating and culturing the rat brain microvascular endothelial cells

Brain microvascular endothelial cells (BMECs), a main component of the blood–brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (>99%) and yield (about 2.2×107 per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood–brain barrier and neurovascular units, etc.

Early exercise training improves ischemic outcome in rats by cerebral hemodynamics

This study examined whether very early initiated physical rehabilitation (VEIPR), as a recommended therapy for postischemia, could improve motor performance and cerebral blood flow (CBF). Adult male rats with ischemic injury caused by middle cerebral artery occlusion (MCAO) were trained to run on a treadmill for 30min per day at 12m/min. Through such exercise training for 3 days, the ischemic rats exhibited increased motor function and decreased infarct volume, as measured by a behavioral score and 2,3,5-triphenyltetrazolium chloride (TTC) staining method, as well as accelerated CBF, as detected with laser speckle imaging (LSI). Furthermore, to determine whether the observed improved CBF provided the protective factor for motor function recovery, we investigated the apoptosis of ischemic rat brain microvascular endothelial cells (rBMECs), which accepted the mechanical force of CBF directly, under flow intervention. The findings indicated that a modest flow decreased cell apoptosis in the ischemic condition and that this effect is magnitude dependent, as excessive flow increased apoptosis.

Baicalin protects rat brain microvascular endothelial cells injured by oxygen-glucose deprivation via anti-inflammation

Baicalin, which is isolated from Scutellariae Radix, has been evidenced to possess several pharmacological effects. The present study focuses on the in vitro protective effect of baicalin on oxygen-glucose deprivation (OGD) injured brain microvascular endothelial cells (BMECs) via anti-inflammation and mechanisms against BMECs damaged by OGD. Cultured primary rat BMECs were exposed to baicalin at the concentrations of 100μM (high dose) and 10μM (low dose) for 6h after a 2h OGD. The effects of baicalin were evaluated in terms of (i) cell viability; (ii) lactate dehydrogenase (LDH) leakage rate; (iii) levels of TNF-α, IL-1β, IL-6 in culture media; (iv) protein expressions of p-MEK6, p-MEK1/2, p-ERK, p-IκBα, NF-κB p65, p-IKKα, p-IKKβ and p-p38; and (v) nuclear translocation of NF-κB p65 and p-IκBα. The results showed that OGD treatment could reduce cell viability, increase LDH leakage rate, increase the levels of TNF-α, IL-1β and IL-6 in the culture media. These effects were suppressed by baicalin with high or low dose. In addition, baicalin could notably down-regulate the phosphorylation of proteins in MAPK signaling pathway such as p-MRK1/2, p-ERK and p-p38. While low dose of baicalin could significantly suppress the phosphorylation of proteins in NF-кB signaling pathway such as p-IKKα, p-IKKβ and p-IκBα. Furthermore, baicalin at 10μM could remarkably inhibit nuclear transcriptional activity triggered via NF-κB p65 and p-IκBα in BMECs. In conclusion, baicalin displays a protective effect on OGD-injured BMECs in vitro by attenuating inflammatory factors via down-regulated the MAPK and NF-κB signaling pathway.

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

Mitochondrial Mechanisms That Regulate Blood‐Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen‐Glucose Deprivation and Reoxygenation

Ischemia‐reperfusion (IR) injury that occurs following ischemic stroke and traumatic brain injury induces blood‐brain barrier (BBB) breakdown, hyperpermeability and brain edema. Our objective was to determine the mitochondrial mechanisms that regulate BBB integrity and permeability using an oxygen‐glucose deprivation and reoxygenation (OGD‐R) in vitro model of IR injury. Rat brain microvascular endothelial cell (RBMECs) monolayers grown on Transwell inserts were exposed to OGD‐R for 1 or 3 hours alone or in presence of a caspase‐3 inhibitor Z‐DEVD or L‐ascorbic acid 1 hr prior to OGD or a caspase‐3 siRNA 48 hrs prior to OGD. Monolayer permeability was studied based on FITC‐dextran permeability. RBMECs were exposed to OGD‐R for 1 or 3 hours alone or in presence of L‐ascorbic acid. ROS was measured using DCFDA, cytosolic cytochrome c by ELISA and caspae‐3 activity fluorometrically. OGD‐R for 1 or 3 hours increased monolayer permeability, ROS, cytochrome c levels and caspase‐3 activity (p<0.05). Z‐DEVD, caspase‐3 siRNA and L‐ascorbic acid attenuated OGD‐R induced hyperpermeability (P<0.05). L‐ascorbic acid but not Z‐DEVD decreased ROS formation and cytochrome c levels (p<0.05). Immunofluorescence of ZO‐1 demonstrated disorganization of the TJs and Z‐DEVD and L‐ascorbic acid provided protection. These results suggest a major role for mitochondrial ROS in regulating OGD‐R induced BBB TJ integrity and permeability.

Metformin induces up-regulation of blood–brain barrier functions by activating AMP-activated protein kinase in rat brain microvascular endothelial cells

Blood–brain barrier (BBB) disruption occurs frequently in CNS diseases and injuries. Few drugs have been developed as therapeutic candidates for facilitating BBB functions. Here, we examined whether metformin up-regulates BBB functions using rat brain microvascular endothelial cells (RBECs). Metformin, concentration- and time-dependently increased transendothelial electrical resistance of RBEC monolayers, and decreased RBEC permeability to sodium fluorescein and Evans blue albumin. These effects of metformin were blocked by compound C, an inhibitor of AMP-activated protein kinase (AMPK). AMPK stimulation with an AMPK activator, AICAR, enhanced BBB functions. These findings indicate that metformin induces up-regulation of BBB functions via AMPK activation.

Curcumin Ameliorates the Permeability of the Blood–Brain Barrier During Hypoxia by Upregulating Heme Oxygenase-1 Expression in Brain Microvascular Endothelial Cells

Curcumin (Cur) is a major active component of the food flavor turmeric isolated from the powdered dry rhizome of Curcuma longa Linn., which has been used in traditional Chinese medicine to ameliorate intracerebral ischemic damage and reduce brain edema. However, the effects of Cur on the disruption of the blood-brain barrier (BBB) induced by brain ischemia are still unclear. The effects of Cur on the disruption of BBB and changes of tight junction (TJ) proteins induced by oxygen glucose deprivation (OGD) were studied in BBB in vitro. The transendothelial electrical resistance and the flux of horseradish peroxidase in BBB in vitro were measured. The expression and localization of the TJ proteins occludin and zonula occludens-1 (ZO-1) were evaluated by Western blots and immunofluorescence microscopy. The protein levels of heme oxygenase-1 (HO-1) were also analyzed via Western blots. Cur attenuated OGD-induced disruption of paracellular permeability and increased the expression of HO-1 protein in rat brain microvascular endothelial cells (RBMECs). After administration of OGD, the expression of occludin and ZO-1 proteins was restored by Cur, and this effect was blocked by a HO-1 inhibitor, zinc protoporphyrin (ZnPP). Cur protects RBMECs against OGD-induced disruption of TJ and barrier dysfunction via the HO-1 pathway. We propose that Cur is capable of improving the barrier function of BBB under ischemic conditions and this beneficial effect might be reversed by a HO-1 inhibitor, ZnPP.

Vasoactive intestinal peptide increases VEGF expression to promote proliferation of brain vascular endothelial cells via the cAMP/PKA pathway after ischemic insult in vitro

Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.

Depolarization of Mitochondria in Endothelial Cells Promotes Cerebral Artery Vasodilation by Activation of Nitric Oxide Synthase

Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Shear Stress Inhibits Apoptosis of Ischemic Brain Microvascular Endothelial Cells

As a therapeutic strategy for ischemic stroke, to restore or increase cerebral blood flow (CBF) is the most fundamental option. Laminar shear stress (LS), as an important force generated by CBF, mainly acts on brain microvascular endothelial cells (BMECs). In order to study whether LS was a protective factor in stroke, we investigated LS-intervented ischemic apoptosis of rat BMECs (rBMECs) through PE Annexin V/7-AAD, JC-1 and Hoechst 33258 staining to observe the membranous, mitochondrial and nuclear dysfunction. Real-time PCR and western blot were also used to test the gene and protein expressions of Tie-2, Bcl-2 and Akt, which were respectively related to maintain membranous, mitochondrial and nuclear norm. The results showed that LS could be a helpful stimulus for ischemic rBMECs survival. Simultaneously, membranous, mitochondrial and nuclear regulation played an important role in this process.

Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells

The aim of this study was to examine whether flavin-containing monooxygenase (FMO) protein was expressed in cultured rat brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), and whether N-oxide from the tertiary amine, d-chlorpheniramine, was formed by FMO in rat BMECs. BMECs were isolated and cultured from the brains of three-week-old male Wistar rats. The expression of FMO1, FMO2 and FMO5 proteins was confirmed in rat BMECs by western blotting analysis using polyclonal anti-FMO antibodies, but FMO3 and FMO4 proteins were not found in the rat BBB. Moreover, N-oxide of d-chlorpheniramine was formed in rat BMECs. The intrinsic clearance value for N-oxidation at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent Ki value of 0.53 μmol/L, suggesting that N-oxidation was catalyzed by FMOs in rat BMECs. Although FMO activity in rat BMECs was lower than that in SD rat normal hepatocytes (rtNHeps), we suggest that rat BMECs enzymes can convert substrates of exogenous origin for detoxification, indicating that BMECs are an important barrier for metabolic products besides hepatic cells.

A modular approach to create a neurovascular unit-on-a-chip

In this work, we describe the fabrication and working of a modular microsystem that recapitulates the functions of the "Neurovascular Unit". The microdevice comprised a vertical stack of a poly(dimethylsiloxane) (PDMS) neural parenchymal chamber separated by a vascular channel via a microporous polycarbonate (PC) membrane. The neural chamber housed a mixture of neurons (~4%), astrocytes (~95%), and microglia (~1%). The vascular channel was lined with a layer of rat brain microvascular endothelial cell line (RBE4). Cellular components in the neural chamber and vascular channel showed viability (>90%). The neural cells fired inhibitory as well as excitatory potentials following 10 days of culture. The endothelial cells showed diluted-acetylated low density lipoprotein (dil-a-LDL) uptake, expressed von Willebrand factor (vWF) and zonula occludens (ZO-1) tight junctions, and showed decreased Alexafluor™-conjugated dextran leakage across their barriers significantly compared with controls (p < 0.05). When the vascular layer was stimulated with TNF-α for 6 h, about 75% of resident microglia and astrocytes on the neural side were activated significantly (p < 0.05 compared to controls) recapitulating tissue-mimetic responses resembling neuroinflammation. The impact of this microsystem lies in the fact that this biomimetic neurovascular platform might not only be harnessed for obtaining mechanistic insights for neurodegenerative disorders, but could also serve as a potential screening tool for central nervous system (CNS) therapeutics in toxicology and neuroinfectious diseases.

Role of ROS/RhoA/PI3K/PKB Signaling in NS1619-Mediated Blood–Tumor Barrier Permeability Increase

The calcium-activated potassium channel (K (Ca) channel) activator, NS1619, has been shown to selectively and time-dependently increase the permeability of the blood-tumor barrier (BTB) by downregulating the expression of tight junction (TJ) protein. However, the role of signaling cascades in this process has not been precisely elucidated. This study was performed to determine the role of signaling cascades involving reactive oxygen species (ROS)/RhoA/PI3K/PKB in increasing the permeability of the BTB induced by NS1619. Using an in vitro BTB model and selective inhibitors of signaling pathways, we investigated whether ROS/RhoA/PI3K/PKB pathway plays a key role in the process of the increase in BTB permeability induced by NS1619. The results revealed that the BTB permeability was increased and the expression of TJ proteins were significantly decreased by NS1619, and selective inhibitors of identified signaling pathways reversed the observed alterations. Moreover, the significant increases in ROS, RhoA activity, and PKB phosphorylation after NS1619 administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine or C3 exoenzyme or LY294002 pretreatment. The present study demonstrates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in rat brain microvascular endothelial cells was required for the increase in BTB permeability induced by NS1619.