Rat Brain Microvascular Endothelial Cells Research Articles

Fingolimod (FTY720-P) Does Not Stabilize the Blood–Brain Barrier under Inflammatory Conditions in an in Vitro Model

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P1). Fingolimod phosphate (FTY720-P) a functional S1P1 antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability—in particular, on the tight junction proteins occludin, claudin 5 and ZO-1—has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P1 plays a dual role in vascular permeability, depending on its ligand. Thus, S1P1 provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers—such as the blood-retinal barrier—which might result in macular edema.

Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

Borneol Depresses P-Glycoprotein Function by a NF-κB Signaling Mediated Mechanism in a Blood Brain Barrier in Vitro Model

P-glycoprotein (P-gp) on brain microvascular endothelial cells (BMECs) that form the blood brain barrier (BBB), influences transportation of substances between blood and brain. The objective of this study was to characterize the effects of borneol on P-gp efflux function on BBB and explore the potential mechanisms. We established an in vitro BBB model comprised of rat BMECs and astrocytes to measure the effects of borneol on the known P-gp substrates transport across BBB, and examined the function and expression of P-gp in BMECs and the signaling pathways regulating P-gp expression. Borneol increased intracellular accumulation of Rhodamine 123, enhanced verapamil and digoxin across the BBB in vitro model, and depressed mdr1a mRNA and P-gp expression. Borneol could activate nuclear factor-κB (NF-κB) and inhibition of NF-κB with MG132 (carbobenzoxy-Leu-Leu-leucinal) and SN50 (an inhibitory peptide) obscuring the P-gp decreases induced by borneol. These data suggested that borneol depresses P-gp function in BMECs by a NF-κB signaling medicated mechanism in a BBB in vitro model.

Brain protection against ischemic stroke using choline as a new molecular bypass treatment.

To determine whether administration of choline could attenuate brain injury in a rat model of ischemic stroke and the underlying mechanisms. A rat model of ischemic stroke was established through permanent middle cerebral artery occlusion (pMCAO). After the surgery, the rats were treated with choline or choline plus the specific α7 nAChR antagonist methyllycaconitine (MLA), or with the control drug nimodipine for 10 days. The neurological deficits, brain-infarct volume, pial vessel density and the number of microvessels in the cortex were assessed. Rat brain microvascular endothelial cells (rBMECs) cultured under hypoxic conditions were used in in vitro experiments. Oral administration of choline (100 or 200 mg·kg(-1)·d(-1)) or nimodipine (20 mg·kg(-1)·d(-1)) significantly improved neurological deficits, and reduced infarct volume and nerve cell loss in the ischemic cerebral cortices in pMCAO rats. Furthermore, oral administration of choline, but not nimodipine, promoted the pial arteriogenesis and cerebral-cortical capillary angiogenesis in the ischemic regions. Moreover, oral administration of choline significantly augmented pMCAO-induced increases in the expression levels of α7 nAChR, HIF-1α and VEGF in the ischemic cerebral cortices as well as in the serum levels of VEGF. Choline-induced protective effects were prevented by co-treatment with MLA (1 mg·kg(-1)·d(-1), ip). Treatment of rBMECs cultured under hypoxic conditions in vitro with choline (1, 10 and 100 μmol/L) dose-dependently promoted the endothelial-cell proliferation, migration and tube formation, as well as VEGF secretion, which were prevented by co-treatment with MLA (1 μmol/L) or by transfection with HIF-1α siRNA. Choline effectively attenuates brain ischemic injury in pMCAO rats, possibly by facilitating pial arteriogenesis and cerebral-cortical capillary angiogenesis via upregulating α7 nAChR levels and inducing the expression of HIF-1α and VEGF.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Increases Blood-Tumor Barrier Permeability by Activating the RhoA/ROCK/PI3K Signaling Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can increase blood-tumor barrier (BTB) permeability via both paracellular and transcellular pathways. In addition, we revealed that the RhoA/Rho kinase (ROCK) signaling pathway is involved in EMAP-II-induced BTB opening. This study further investigated the exact mechanisms by which the RhoA/ROCK signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II significantly activated phosphatidylinositol-3-kinase (PI3K) in rat brain microvascular endothelial cells (RBMECs) at 0.75h. Pretreatment with RhoA inhibitor C3 exoenzyme or ROCK inhibitor Y-27632 completely blocked EMAP-II-induced activation of PI3K. PKC-α/β inhibitor GÖ6976 pretreatment caused no change in EMAP-II-induced activation of PI3K. Besides, pretreatment with LY294002, a specific inhibitor of PI3K, did not affect EMAP-II-induced activation of PKC-α/β. Furthermore, LY294002 pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, phosphorylation of myosin light chain and cofilin, expression and distribution of tight junction-associated protein ZO-1, and actin cytoskeleton arrangement in RBMECs. In summary, this study demonstrates that low-dose EMAP-II can increase BTB permeability by activating the RhoA/ROCK/PI3K signaling pathway.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Induces Blood-Tumor Barrier Opening Via the cAMP/PKA/Rac1 Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. This study further investigated the exact mechanisms through which the cAMP/PKA-dependent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rac1 activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced inactivation of Rac1. Besides, pretreatment with 6Bnz-cAMP to activate PKA partially attenuated EMAP-II-induced Rac1 inactivation. Moreover, 6Bnz-cAMP pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, myosin light chain (MLC) phosphorylation, expression and distribution of ZO-1, and actin cytoskeleton arrangement in RBMECs. These effects of 6Bnz-cAMP were completely blocked in the presence of NSC-23766 (the specific inhibitor of Rac1). In conclusion, this study demonstrates that low-dose EMAP-II induces BTB hyperpermeability via the cAMP/PKA/Rac1 signaling pathway.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

BackgroundSilver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood.MethodTo investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray.ResultsA triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes.ConclusionThese results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers.

The blood brain barrier (BBB) is often an insurmountable obstacle for a large number of candidate drugs, including peptides, antibiotics, and chemotherapeutic agents. Devising an adroit delivery method to cross the BBB is essential to unlocking widespread application of peptide therapeutics. Presented here is an engineered nanocontainer for delivering peptidic drugs across the BBB encapsulating the analgesic marine snail peptide ziconotide (Prialt®). We developed a bi-functional viral nanocontainer based on the Salmonella typhimurium bacteriophage P22 capsid, genetically incorporating ziconotide in the interior cavity, and chemically attaching cell penetrating HIV-Tat peptide on the exterior of the capsid. Virus like particles (VLPs) of P22 containing ziconotide were successfully transported in several BBB models of rat and human brain microvascular endothelial cells (BMVEC) using a recyclable noncytotoxic endocytic pathway. This work demonstrates proof in principle for developing a possible alternative to intrathecal injection of ziconotide using a tunable VLP drug delivery nanocontainer to cross the BBB.

Roles of Serine/Threonine Phosphatases in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood–Tumor Barrier

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) opening via RhoA/Rho kinase/PKC-α/β signaling pathway. In a recent study, we revealed that low-dose EMAP-II induced significant increases in expression levels of serine/threonine (Ser/Thr) phosphatase (PP)1 and 2A in rat brain microvascular endothelial cells (RBMECs) of BTB model. In addition, PKC-ζ/PP2A signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact roles of PPs in this process. In an in vitro BTB model, low-dose EMAP-II (0.05nM) induced a significant increase in PP1 activity in RBMECs. There was an interaction between PKC-α/β and PP1 in RBMECs. Inhibition of PKC-α/β activity with GÖ6976 completely blocked EMAP-II-induced activation of PP1. Conversely, inhibition of PP1 activity with tautomycin had no effect on EMAP-II-induced PKC-α/β activation. Like GÖ6976, tautomycin significantly prevented EMAP-II-induced BTB hyperpermeability and MLC phosphorylation in RBMECs. Also, in this study, EMAP-II induced a marked redistribution of occludin and a significant dephosphorylation of occludin on Ser/Thr residues in RBMECs. Similar with GÖ6976 pretreatment, tautomycin pretreatment dramatically diminished EMAP-II-induced redistribution of occludin. Furthermore, pretreatment with tautomycin significantly inhibited EMAP-II-induced dephosphorylation of occludin on Ser residues. However, pretreatment with okadaic acid (an inhibitor of PP2A) significantly prevented changes in Ser-phosphorylated occludin induced by EMAP-II treatment. Collectively, this study demonstrates that low-dose EMAP-II increases BTB permeability via a RhoA/Rho kinase/PKC-α/β/PP1 signaling pathway and that PP1/PP2A-mediated Ser/Thr dephosphorylation of occludin plays an important role in EMAP-II-induced BTB hyperpermeability.

Brain pericytes are the most thrombin-sensitive matrix metalloproteinase-9-releasing cell type constituting the blood–brain barrier in vitro

In the acute phase of intracerebral hemorrhage (ICH), hemorrhagic transformation and brain edema are associated with blood–brain barrier (BBB) disruption. Elevated levels of thrombin, a coagulation factor, contribute to the development of brain edema during ICH through matrix metalloproteinase (MMP)-9 production. Thrombin directly induces a variety of cellular responses through its specific receptors known as protease-activated receptors (PARs). However, it remains unclear which cell types constituting the BBB mainly produce MMP-9 in response to thrombin. Here, we compared the MMP-9 release induced by thrombin using primary cultures of rat brain microvascular endothelial cells, astrocytes, and pericytes. Brain pericytes exhibited the highest levels of MMP-9 release due to thrombin stimulation among the BBB cells. The pattern of PAR mRNA expression in pericytes was characterized by high expression of PAR1 and moderate expression of PAR4. Heat-inactivated thrombin failed to stimulate pericytes to release MMP-9. A selective PAR1 inhibitor SCH79797 blocked the thrombin-induced MMP-9 release from pericytes. These findings suggest that both PAR1 and PAR4 mediate thrombin-induced MMP-9 release from pericytes. The present study raises the possibility that brain pericytes could play a pivotal role as a highly thrombin-sensitive and MMP-9-producing cell type at the BBB in brain damage including ICH.

Pretreatment with docosahexaenoic acid enhances the protective effect of angiopoietin-1 on rat brain microvascular endothelial cells during oxygen glucose deprivation

Objective To evaluate the role of pretreatment with docosahexaenoic acid (DHA) in the protective effect of angiopoietin-1 (Ang-1) on rat brain microvascular endothelial cells (BMVECs) during oxygen glucose deprivation (OGD). Methods BMVECs were sub-cultured in vitro and divided with a random number table into 7 groups: normal control group, normal control+ Ang-1 group, OGD group, OGD+ Ang-1 group, OGD+ DHA group, OGD+ DHA+ Ang-1 group, and OGD+ DHA+ GW9662+ Ang-1 group. The normal control and normal control+ Ang-1 groups were cultured in DMEM containing serum, 5 mmol/L glucose, and 1.25 mmol/L pyruvate; OGD groups were cultured in glucose- and serum-free DMEM. DHA (40 μmol/L) was added to OGD+ DHA, OGD+ DHA+ Ang-1, and OGD+ DHA+ GW9662+ Ang-1 groups, and 5 μmol/L GW9662 [inhibitor of peroxisome proliferator-activated receptor gamma (PPAR-γ)] to OGD+ DHA+ GW9662+ Ang-1 group, before pretreatment for 1 hour in 5% CO2 and 95% air. After the pretreatment, Ang-1 (250 ng/ml) was added to normal control+ Ang-1, OGD+ Ang-1, OGD+ DHA+ Ang-1, and OGD+ DHA+ GW9662+ Ang-1 groups. The cells were cultured in 94% N2∶5% CO2∶1%O2 for 24 hours, except for normal control and normal control+ Ang-1 groups, which were cultured in 5% CO2 and 95% air instead. The cell apoptosis rate was detected with flow cytometry, expressions of Bax, Bcl-2, caspase-3, Tie-1, Tie-2, pTie-2, pAkt, ZO-1 proteins with Western blot. Results Compared with normal control group, the cell apoptosis rate in OGD, OGD+ Ang-1, OGD+ DHA, OGD+ DHA+ Ang-1, and OGD+ DHA+ GW9662+ Ang-1 groups were significantly increased (P=0.000, 0.000, 0.000, 0.004, 0.000); the expression levels of Bax, caspase-3, and Tie-1 were significantly enhanced (Bax: 0.62±0.03, 0.38±0.03, 0.45±0.03, 0.26±0.02, 0.33±0.02 vs. 0.16±0.01; caspase-3: 0.76±0.05, 0.42±0.04, 0.52±0.02, 0.32±0.02, 0.40±0.02 vs. 0.15±0.01; Tie-1: 0.51±0.03, 0.25±0.01, 0.33±0.02, 0.16±0.01, 0.22±0.02 vs. 0.12±0.01; all P=0.000); the expression levels of Bcl-2, Bcl-2/Bax, Tie-2, and pTie-2 were significantly decreased [Bcl-2: 0.09±0.01, 0.20±0.01, 0.16±0.02, 0.31±0.01, 0.22±0.01 vs. 0.34±0.01; Bcl-2/Bax: (14.93±1.86)%, (68.03±5.56)%, (36.93±2.22)%, (119.1±13.3)%, (64.23±6.07)% vs. (208.33±7.37)%; Tie-2: 0.07±0.01, 0.16±0.02, 0.11±0.01, 0.21±0.01, 0.18±0.01 vs. 0.26±0.01; pTie-2: 0.05±0.01, 0.15±0.01, 0.07±0.01, 0.22±0.02, 0.16±0.01 vs. 0.27±0.01; all P=0.000], in addition, pAkt and ZO-1 in OGD, OGD+ Ang-1, OGD+ DHA, and OGD+ DHA+ GW9662+ Ang-1 groups were also significantly reduced (pAkt: 0.13±0.01, 0.26±0.01, 0.14±0.01, 0.28±0.02 vs. 0.39±0.02; ZO-1: 0.08±0.01, 0.18±0.01, 0.10±0.01, 0.19±0.01 vs. 0.23±0.02; all P=0.000). Compared with the OGD group, OGD+ Ang-1 and OGD+ DHA+ Ang-1 groups demonstrated significantly reduced cell apoptosis (both P=0.000). Compared with normal control+ Ang-1 group, the expression levels of pTie-2, pAkt, and ZO-1 were significantly decreased in OGD+ Ang-1 and OGD+ DHA+ Ang-1 groups (pTie-2: 0.15±0.01, 0.22±0.02 vs. 0.52±0.02; pAkt: 0.26±0.01, 0.37±0.04 vs. 0.67±0.05; ZO-1: 0.18±0.01, 0.24±0.02 vs. 0.39±0.05; all P=0.000). Compared with OGD+ Ang-1 group, OGD+ DHA+ Ang-1 group had significantly decreased cell apoptosis and expression levels of Bax, caspase-3 and Tie-1, and significantly increased expressions of Bcl-2, Bcl-2/Bax, Tie-2, pTie-2, pAkt, and ZO-1 (all P=0.000), while OGD+ DHA+ GW9662+ Ang-1 group showed no significant differences in these indexes (P=0.202, 0.770, 0.382, 0.448, 0.233, 0.736, 0.143, 0.526, 0.495, 0.670). Conclusion Pretreatment with DHA may enhance the protective effect of Ang-1 on rat BMVECs under the condition of OGD, possibly via activating PPAR-γ, suppressing Tie-1 expression, and hence amplifying the activation of Tie-2. Key words: Docosahexaenoid acid; Oxygen glucose deprivation; Cell apoptosis; Angiopoietin-1; Brain microvascular endothelial cells

PJ-34 inhibits PARP-1 expression and ERK phosphorylation in glioma-conditioned brain microvascular endothelial cells

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD+, the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

G protein-coupled estrogen receptor-mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells.

G protein-coupled estrogen receptor (GPER) is a relatively recently identified non-nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca(2+) concentration, [Ca(2+)](i), nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G-1, a selective GPER agonist, or extracellular administration of 17-β-estradiol and tamoxifen, increased [Ca(2+)](i) in RBMVEC. The effect of G-1 on [Ca(2+)](i) was abolished in Ca(2+) -free saline or in the presence of a L-type Ca(2+) channel blocker. G-1 increased nitric oxide production in RBMVEC; the effect was prevented by NG-nitro-l-arginine methyl ester. G-1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate-conductance Ca(2+) -activated K(+) channels, apamin, and charibdotoxin. GPER-mediated responses were sensitive to G-36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G-1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature. Activation of the G protein-coupled estrogen receptor (GPER) in rat brain microvascular endothelial cells (RBMVEC) increases [Ca(2+)](i) by promoting Ca(2+) influx. The increase in [Ca(2+)](i) leads to membrane hyperpolarization, nitric oxide (NO) production, and to cytoskeletal changes and increased cell stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

Low-dose endothelial monocyte-activating polypeptide-II increases permeability of blood–tumor barrier via a PKC-ζ/PP2A-dependent signaling mechanism

Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood–tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/β signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function.

Role of cAMP-dependent protein kinase A activity in low-dose endothelial monocyte-activating polypeptide-II-induced opening of blood-tumor barrier.

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Tumor necrosis factor-α disruption of brain endothelial cell barrier is mediated through matrix metalloproteinase-9

Traumatic brain injuries cause vascular hyperpermeability. Tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), and caspase-3 may be important in these processes but the relationship between them has not been investigated. We hypothesized that TNF-α regulates caspase-3-mediated hyperpermeability and blood brain barrier damage and hyperpermeability directly or indirectly via activation of MMP-9. To test this, rat brain microvascular endothelial cells were treated with TNF-α with or without inhibition of MMP-9. Monolayer permeability was measured, zonula occludens-1 and F-actin configuration were examined, and MMP-9 and caspase-3 activities were quantified. TNF-α increased monolayer permeability, damaged zonula occludens-1, induced filamentous-actin stress fiber formation, and increased both MMP-9 and caspase-3 activities. Inhibition of MMP-9 attenuated these changes. These data highlight a novel link between TNF-α and MMP-9 and show that TNF-α regulated caspase-3-mediated hyperpermeability and vascular damage may be linked to MMP-9 in vitro. These findings augment the understanding of traumatic brain injury and pave the way for improved treatment.

Rhythmic expression of cytochrome P450 epoxygenases CYP4x1 and CYP2c11 in the rat brain and vasculature.

Mammals have circadian variation in blood pressure, heart rate, vascular tone, thrombotic tendency, and cerebral blood flow (CBF). These changes may be in part orchestrated by circadian variation in clock gene expression within cells comprising the vasculature that modulate blood flow (e.g., fibroblasts, cerebral vascular smooth muscle cells, astrocytes, and endothelial cells). However, the downstream mechanisms that underlie circadian changes in blood flow are unknown. Cytochrome P450 epoxygenases (Cyp4x1 and Cyp2c11) are expressed in the brain and vasculature and metabolize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs). EETs are released from astrocytes, neurons, and vascular endothelial cells and act as potent vasodilators, increasing blood flow. EETs released in response to increases in neural activity evoke a corresponding increase in blood flow known as the functional hyperemic response. We examine the hypothesis that Cyp2c11 and Cyp4x1 expression and EETs production vary in a circadian manner in the rat brain and cerebral vasculature. RT-PCR revealed circadian/diurnal expression of clock and clock-controlled genes as well as Cyp4x1 and Cyp2c11, within the rat hippocampus, middle cerebral artery, inferior vena cava, hippocampal astrocytes and rat brain microvascular endothelial cells. Astrocyte and endothelial cell culture experiments revealed rhythmic variation in Cyp4x1 and Cyp2c11 gene and protein expression with a 12-h period and parallel rhythmic production of EETs. Our data suggest there is circadian regulation of Cyp4x1 and Cyp2c11 gene expression. Such rhythmic EETs production may contribute to circadian changes in blood flow and alter risk of adverse cardiovascular events throughout the day.

Amyloid-beta disrupts calcium and redox homeostasis in brain endothelial cells.

In Alzheimer's disease, the accumulation of amyloid-beta (Aβ) in the brain occurs in the parenchyma and cerebrovasculature. Several evidences support that the neuronal demise is potentiated by vascular alterations in the early stages of the disease, but the mechanisms responsible for the dysfunction of brain endothelial cells that underlie these cerebrovascular changes are unknown. Using rat brain microvascular endothelial cells, we found that short-term treatment with a toxic dose of Aβ1-40 inhibits the Ca(2+) refill and retention ability of the endoplasmic reticulum and enhances the mitochondrial and cytosolic response to adenosine triphosphate (ATP)-stimulated endoplasmic reticulum Ca(2+) release. Upon prolonged Aβ1-40 exposure, Ca(2+) homeostasis was restored concomitantly with a decrease in the levels of proteins involved in its regulation operating at the plasma membrane, endoplasmic reticulum, and mitochondria. Along with perturbations in Ca(2+) regulation, an early increase in the levels of oxidants and a decrease in the ratio between reduced and oxidized glutathione were observed in Aβ1-40-treated endothelial cells. Under these conditions, the nuclear levels of oxidative stress-related transcription factors, namely, hypoxia-inducible factor 1α and nuclear factor (erythroid-derived 2)-related factor 2, were enhanced as well as the protein levels of target genes. In conclusion, Aβ1-40 affects several mechanisms involved in Ca(2+) homeostasis and impairs the redox homeostasis simultaneously with stimulation of protective stress responses in brain endothelial cells. However, the imbalance between cell death and survival pathways leads to endothelial dysfunction that in turn contributes to cerebrovascular impairment in Alzheimer's disease.

Inhibition of endothelin A receptor protects brain microvascular endothelial cells against hypoxia‑induced injury.

Endothelin-1 (ET-1)-induced cell damage is commonly involved in ischemia/hypoxia-associated diseases. PD155080 [sodium 2-benzo (1.3)dioxol-5-yl-3-benzyl-4-(4‑metho-xyphenyl)-4-oxobut-2-enoate] is a selective endothelin A receptor (ETAR) antagonist that inhibits ET-1‑induced cell damage. The aim of this study was to investigate the effects of PD155080 on hypoxia-induced rat brain microvascular endothelial cell (BMEC) injury. BMECs were isolated from the cerebral cortex of Wistar rats and cultured in an anoxia chamber, containing 95% N(2) and 5% CO(2) for 12 h. BMEC injury was assessed by determining cellular ultra-microstructural changes and cell viability by MTT assay, trypan blue (TB) staining and measuring the lactate dehydrogenase (LDH) levels. ET-1 mRNA expression was detected by in situ hybridization and reverse transcription PCR (RT-PCR); the ET-1 protein level was measured by radioimmunoassay. Following exposure to hypoxic conditions, the viability of the BMECs was markedly decreased and the ultrastructure of the BMECs was damaged, as demonstrated by chromatin margination, chromatin agglutination, plasma edema, the increased number of intracellular liposomes and vacuoles, mitochondrial swelling and the expansion of a rough surfaced endoplasmic reticulum. The levels of ET-1 and ET-1 mRNA expression in the BMECs were increased following exposure to hypoxic conditions. Of note, the administration of PD155080 greatly enhanced the viability of the BMECs and ameliorated hypoxia-induced cellular injury. PD155080 also inhibited hypoxia-induced ET-1 production by the BMECs. In conclusion, PD155080 exerts protective effects against hypoxia-induced BMEC injury.

The high-mobility group I-Y transcription factor is involved in cerebral ischemia and modulates the expression of angiogenic proteins

The present study aims to identify transcription factors (TFs) contributing to angiogenesis, a mechanism involved in giving plasticity to the brain, as potential therapeutic targets after cerebral ischemia. The promoter sequences from candidate genes involved in angiogenesis were submitted to a comparative analysis by bioinformatics software. High-mobility group I-Y protein (HMGIY) TF characterization in a rat permanent focal cerebral ischemia model was performed by quantitative real time polymerase chain reaction and Western blot for the TF expression profile study. The TF functional study was carried out using a TF–TF interaction array and gene silencing by siRNA in rat brain microvascular endothelial cells. The results showed that the promoters shared a common TF binding site for HMGIY. The expression profile analysis in ischemic rat brain showed an increase in HMGIY mRNA in the acute phase and a progressive overexpression of protein over time post-ischemia. The interaction array analysis revealed that ischemia promotes the interaction of HMGIY with TFs involved in different cerebral plasticity processes. In vitro knockdown studies showed that angiopoietin 1 and vascular endothelial growth factor expression is controlled by HMGIY and that this TF is involved in cell survival in brain endothelial cells. These findings suggest that HMGIY is a potential therapeutic target that could promote brain repair functions after stroke.