Midazolam Inhibits Tumor Necrosis Factor-α–induced Endothelial Activation: Involvement of the Peripheral Benzodiazepine Receptor

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (Cambrex, MD), and they were grown and maintained in endothelial growth medium. Cells were used between passages 3 and 6. U937 cell lines were obtained from American type culture collection (Manassas, VA). Midazolam was purchased from Bukwang Company (Seoul, Korea). Midazolam was dissolved in 0.9% NaCl solution that was used as vehicle for treatment of midazolam.10,11For the induction of endothelial cell activation, human TNF-α (15 ng/ml) was used. Anti-VCAM-1 and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PBR was from Novus Biologicals. Horseradish peroxidase-labeled anti-rabbit antibodies were from Amersham (Buckinghamshire, United Kingdom). Human TNF-α, PK11195, 1-(2-chlorophenyl)-N -methyl-N -(1-methyl-propyl)-3-isoquinoline carboxamide, and flumazenil were purchased from Sigma (St. Louis, MO). PK11195 was dissolved with ethanol as a vehicle and then diluted in the endothelial growth medium-2 (Cambrex, MD). Maximal concentration of vehicle (ethanol) for PK11195 was 0.01%. To inhibit benzodiazepine receptors, the endothelial cells were pretreated with PK11195 and flumazenil 1 h before treatment of midazolam.

The primers with the restriction enzyme linkers, sense primer: 5′-CCA AGC TTA TGG CCC CGC CCT GGG TG -3′, antisense primer: 5′-CGG AAT TCC CTC TGG CAG CCG CCG TCC-3′, were used for PCR amplification of hPBR. The nucleotide sequencing of PCR products contained an identical coding sequence of hPBR (510 bp) (Genbank accession number BC001110). PBR-EGFP plasmid was generated by inserting hPBR gene into Hind  III and Eco  RI sites of pEGFP-N1 (Clontech, Mountain View, CA).

For transfection and immunofluorescent staining, 5 × 10⁴cells of HUVEC cells were grown on glass coverslips and then transiently transfected with 1 μg of pPBR-EGFP by using Effectene, as recommended by the manufacturer (Qiagen, Valencia, CA). 24 h after transfection, 10 nM of Mitotracker Red CNXRos (Invitrogen, Carlsbad, CA) for 1 h was used for staining mitochondria. Coverslips were mounted on microscope slides, and fluorescence signals were visualized with an Olympus confocal microscope.

Small interfering RNAs (siRNAs) to human PBR consisting of 21 nucleotides were synthesized from Bioneer Co. (Daejeon, Korea). The GenBank accession number for human PBR is NM_000714. siRNA duplexes with the following gene-specific sense sequences were used: PBR 579 siRNA targeting human PBR exon, 5′-CCA UGG CUG GCG UGG GGG AdTdT-3′; PBR 564 siRNA targeting human PBR exon, 5′-CGU AUG GCG GGA CAA CCA UdTdT-3′; PBR 549 siRNA targeting human PBR exon, 5′-CAC ACU CAA CUA CUG CGU A-3′; PBR 788 siRNA targeting human PBR exon, 5′-CCU GUG CUU UCU GCA UGC U-3′. The negative control siRNA (Bioneer Co) was used as control; negative control oligonucleotide template, 5′-CCUACGCCACCAAUUUCGU-3′. A single transfection of 100 nM siRNA duplexes was performed using the Lipofectamine 2000 reagent (Invitrogen). The cells were assayed for silencing later. Cells had been seeded the previous day in the endothelial basal medium-2 supplemented with 2% fetal bovine serum and endothelial growth medium-2 singlequots (Cambrex, Rutherford, NJ). The siRNA duplex was mixed with Opti-MEM. In a separate tube, the Lipofectamine 2000 reagent was mixed with Opti-MEM and incubated for 5 min at room temperature. The two solutions were combined, gently mixed by inversion, and incubated for 20 min at room temperature. The resulting siRNA-Lipofectamine 2000 was added to cells that were cultured at 30–50% confluence. Fresh endothelial growth medium-2 was added to transfected cells 5 h later. Cells were incubated at 37°C in a carbon dioxide incubator for 72 h for gene knockdown.

Monocyte-endothelial cell adhesion assay was performed as described previously.10,12U937 cells were fluorescently labeled with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein acetoxymethyl ester (BCECF-am.) for the quantitative adhesion assay. The U937 cells were fluorescently labeled by incubating the cells (1 × 10⁷cells/ml) with 1 μM BCECF-AM in RPMI-1640 medium for 30 min at 37°C and 5% CO². HUVECs were seeded in 24-well plates to reach confluent monolayers and pretreated with midazolam for 1 h in EGM-2 medium. Human recombinant TNF-α was added to appropriate wells (15 ng/ml) at 18 h before addition of labeled monocytes. Monocyte adhesion was quantified by measuring fluorescence with excitation (485 nm) and emission (535 nm). Wells containing HUVEC only without U937 cells were used as blanks.

For Western blot analysis, HUVECs were harvested with 100 μl of lysis buffer containing 20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM Na³VO³, 1 mM β-glycerophosphate, 4 mM Na pyrophosphate, 5 mM NaF, 1% Triton X-100, and protease inhibitor cocktail. The lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected. Protein (30 μg) was separated by 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. After blocking with 5% skim milk for 2 h at room temperature, blots were incubated overnight at 4°C with specific primary antibody (1:1000), and subsequent detection with horseradish peroxidase–conjugated secondary antibody was performed. Blots were developed for visualization using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL).

Values are expressed as the mean ± SEM. Statistical evaluation was performed using ANOVA analysis followed by post hoc  Tukey with SPSS software (version 14.0, SPSS inc., Chicago, IL), with P < 0.05 considered significant.

We investigated the subcellular localization of PBR by immunofluorescent analysis. After transfection of the cells with pPBR-EGFP, Mitotracker red staining was performed to visualize cellular mitochondria. As shown in figure 1, both mitochondria (red fluorescence with MitoTracker Red) and PBR-EGFP (green fluorescence) presented perinuclear localization. When the images were merged, a colocalization between mitochondria and PBR was clearly observed, confirming a mitochondrial localization of PBR.

To explore whether midazolam regulates VCAM-1 expression, we examined the effect of midazolam on the VCAM-1 expression in the TNF-α–stimulated HUVECs. After pretreatment with midazolam at 1–30 μM, the cells were exposed to TNF-α (15 ng/ml) for 18 h. The pretreatment with midazolam suppressed TNF-α-induced VCAM-1 expression in a dose-dependent manner (1–30 μM; fig. 2, A and B). Since 50% inhibition dose (ID⁵⁰) of midazolam on VCAM-1 is about 5 μM, this dose was used to examine the effect of antagonists on midazolam-mediated inhibition.

VCAM-1 mediates the recruitment and adhesion of monocytes in the activated endothelial cells. After the inhibitory action of midazolam in TNF-α–induced VCAM-1 expression was confirmed, we evaluated the possible role of midazolam in TNF-α–induced monocyte adhesion to endothelial cells. Cells were pretreated with midazolam for 1 h before treatment with TNF-α. After incubation with TNF-α for 18 h, monocyte adhesion assay was performed as described in Material and Methods. As shown in figure 3, monocyte adhesion was minimal in unstimulated endothelial cells, but treatment with TNF-α resulted in a marked increase in monocyte adhesion to endothelial cells. Pretreatment with midazolam (3 to 30 μM) inhibited TNF-α–induced monocyte adhesion to endothelial cells. However, midazolam itself did not induce the monocyte adhesion, suggesting that midazolam has specific inhibitory action on monocyte adhesion induced by TNF-α to endothelial cells.

Midazolam is classified as a mixed-type agonist of benzodiazepine receptors according to their affinity to the central and peripheral binding site.13We next studied the involvement of benzodiazepine receptors in the midazolam-mediated suppression of VCAM-1 expression. To test the involvement of benzodiazepine receptors, we used PK11195, an antagonist for the peripheral benzodiazepine receptor, and flumazenil, an antagonist for the benzodiazepine receptor in the CNS. As shown in figure 4, pretreatment with 10 μM flumazenil did not affect the midazolam-mediated suppression of VCAM-1 expression, suggesting that the central benzodiazepine receptor is not involved in the action of midazolam. In contrast, the pretreatment with 3 μM PK11195 significantly inhibited the midazolam-mediated suppression of VCAM-1 expression in HUVEC, suggesting the involvement of PBR in the action of midazolam.

We examined whether the action of midazolam on monocyte adhesion was mediated by the PBR. To test the involvement of benzodiazepine receptors, we used PK11195 and flumazenil. As shown in figure 5, the pretreatment with 10 μM flumazenil did not reverse the action of midazolam on the monocyte adhesion induced by TNF-α. In contrast, the pretreatment with 3 μM PK11195 significantly attenuated the action of midazolam on the monocyte adhesion induced by TNF-α in HUVEC, suggesting the involvement of PBR in the action of midazolam.

We evaluated the efficiency of siRNAs (100 nM) targeting various PBR coding regions on the PBR expression in the HUVEC. Seventy-two hours of treatment with siRNA (100 nM) targeting various PBR coding regions decreased PBR protein (fig. 6A). Especially, 564 PBR siRNA significantly decreased PBR protein levels. Therefore, 564 PBR siRNA (siRNA #2) was used in the following study.

After treatment of HUVEC cells with 100 nM PBR-specific 564 siRNA (siRNA #2) for 72 h, the inhibitory action of midazolam to TNF-α–induced VCAM-1 expression was significantly suppressed as shown in figure 6, B and C. However, PBR expressions were not affected by the treatment with midazolam and/or TNF-α. These data strongly suggested that the activation of PBR by midazolam was involved in the antiinflammatory action of midazolam in the endothelial cells.

The present study demonstrates that midazolam has an antiinflammatory action against endothelial cell activation. The antiinflammatory action of midazolam was mediated by activating the peripheral benzodiazepine receptor in the endothelial cells. Also, the data provided here indicate that PBR plays a crucial role of antiinflammatory action in endothelial cells.

To confirm intracellular localization of PBR in the present study, we cloned the human PBR cDNA using reverse transcriptase–polymerase chain reaction in HUVECs. Isolated PBR cDNA was completely matched with GenBank accession number BC001110. The human PBR cDNA encodes for a 170–amino acid (18 kDa) protein. Isolated hPBR cDNA was then subcloned into EGFP expressed vectors to study the intracellular localization of hPBR. In 1999, PBR in the endothelial cells was first demonstrated in the dermal vascular endothelial cells.14The putative function of PBR is related to its intracellular localization. In the present study, we confirmed that the PBR of endothelial cells were localized in the mitochondria by using fluorescent imaging for PBR (fig. 1). Mitochondria play a central role in cell function by regulating adenosine triphosphate production and induction of cell death.15A previously published report has shown that the mitochondrial localization of PBR may be involved in the inhibition of mitochondrial respiration.16 

As a benzodiazepine derivative, midazolam is a mixed-type agonist of PBRs, and it is the most widely used anesthetic for sedation. To investigate the role of midazolam in the endothelial cell activation, we used TNF-α to induce endothelial cell activation as we have done in our previous reports.12,17TNF-α is known to cause monocyte adhesion, the expression of adhesion molecules, and marked oxidative stress in the endothelial cells.12 

VCAM-1 gene encodes a cell surface sialoglycoprotein expressed by cytokine-activated endothelium. VCAM-1 protein mediates leukocyte-endothelial cell adhesion and signal transduction, and it may play a role in the development of atherosclerosis. In the present study, we found that midazolam in the range of 3–30 μM suppresses VCAM-1 and monocyte adhesion in the TNF-α–activated endothelial cells. The plasma concentrations of benzodiazepines used clinically were approximately between 0.1 and 50 μM.7,18These data suggest that clinically administered midazolam has antiinflammatory properties through suppression of monocyte adhesion in endothelial cells. Therefore, our data suggest that midazolam, via  peripheral-type benzodiazepine binding sites, may exert an antiinflammatory effect on endothelial cells. Recently, it has been reported that decreased cerebral endothelial intercellular cell adhesion molecule-1 expression by midazolam may decrease post-ischemic brain inflammation and secondary brain injury.19 

We next investigated how midazolam acts on the antiinflammatory action in the endothelial cells. To confirm whether midazolam acts on the PBR or CBR, we used the specific antagonist for PBR or CBR. PK11195 is a widely studied PBR ligand that has been classified as an antagonist of PBR.20–22Flumazenil is a specific and competitive antagonist at the central benzodiazepine receptor23that reverses the effects of benzodiazepines by competitive inhibition at the benzodiazepine binding site on the GABA^Areceptor. Our data show that midazolam-mediated suppression of VCAM-1 and monocyte adhesion was specifically blocked by PK11195, and not by flumazenil, in the endothelial cells. These data suggest that midazolam has an inhibitory action against vascular endothelial cells activation via  the binding of PBR in the endothelial cells. However, midazolam-mediated suppression on monocyte adhesion was only attenuated by PK11195, suggesting that anti-adhesive action of midazolam is not limited by acting in the peripheral benzodiazepine receptor. On the basis of our knowledge, the role of CBR is still unknown in the endothelial cells; even GABA receptors were detected in the endothelial cells (data not shown). The functional role of GABA receptors in the endothelial cells needs further evaluation.

The siRNA targeting PBR was also used to evaluate the role of PBR on the action of midazolam to VCAM-1 expression in the endothelial cells. siRNA duplexes for PBR gene were transfected in the endothelial cells. Seventy-two hours after transfection of siRNA duplexes for PBR, PBR protein expression was markedly decreased as shown in the Western blot against anti-PBR (fig. 6). Midazolam-mediated VCAM-1 suppression was significantly decreased by PBR gene silencing with siRNA duplex transfection in the endothelial cells, suggesting the involvement of PBR on antiinflammatory action.

Part of the signal transduction pathway that regulates the activation of VCAM-1 expression is redox-sensitive; therefore, the compounds with antioxidant properties may have inhibitory effects on VCAM-1 expression.12,24A previous report has shown that midazolam inhibits superoxide production by inhibiting nicotinamide adenine dinucleotide phosphate-oxidase in macrophage.10Midazolam has also been found to inhibit 2,4 dinitrophenol-uncoupled mitochondrial respiration, suggesting that midazolam primarily acts as a mitochondrial electron transport inhibitor.25We therefore proposed that the antiinflammatory action of midazolam is mediated by the binding of PBR. The binding of PBR with midazolam would affect the mitochondrial respiration. Reduced mitochondrial respiration resulted in reduced superoxide production; therefore, mitochondrial superoxide production induced by cytokine such as TNF-α would be suppressed by the midazolam. The endothelial cell activation induced by TNF-α can be reduced by the pretreatment of midazolam.

There are some limitations to our study. First, it was only performed in cultured endothelial cells. The beneficial effect of midazolam on the in vivo  system is limited. Therefore, further studies are required to verify this beneficial effect. Second, regarding the clinical value of midazolam, we acknowledge that monocyte adhesion may be one of several factors contributing to the vascular endothelial activation seen in vascular inflammatory disorder. However, our data are implying a beneficial effect of midazolam in the cytokine-induced vascular inflammation. Another limitation in this study is the pretreatment of midazolam before exposure to cytokine such as TNF-α. The effect of midazolam after exposure to cytokine, like clinical situations, needs further evaluation. Even though this finding implies a beneficial effect in the cytokine-induced vascular inflammation, the clinical application for the vascular inflammation is carefully recommended.

Taken together, our data suggest that midazolam plays an antiinflammatory action in vascular endothelial activation. This antiinflammatory action might be related to binding of the peripheral benzodiazepine receptor expressed in mitochondria of the endothelial cells.