Propofol protects against nitrosative stress-induced apoptotic insults to cerebrovascular endothelial cells via an intrinsic mitochondrial mechanism Ruei-Ming Chen, PhD,a,b,c Yu-Tyng Tai, MD,b Tyng-Guey Chen, MD,b The-Hin Lin, MS,a Huai-Chia Chang, MD, PhD,c Ta-Liang Chen, MD, PhD,c and Gong-Jhe Wu, MD, PhD,a,c,d Taipei, Taiwan
Background. Cerebrovascular endothelial cells (CECs), major component cells of the blood-brain barrier, can be injured by oxidative stress. Propofol can protect cells from oxidative injury. The aim of this study was to evaluate the effects of propofol on nitrosative stress-induced insults to CECs and its possible mechanisms. Methods. Primary CECs isolated from mouse cerebral capillaries were exposed to2 nitric oxide (NO) donors: sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO). Cellular NO levels, cell morphologies, and cell viabilities were analyzed. DNA fragmentation and apoptotic cells were quantified using flow cytometry. Proapoptotic Bcl2-antagonist-killer (Bak) and cytochrome c were immunodetected. Bak translocation was analyzed using confocal microscopy. Caspases-9 and -3 activities were measured fluorometrically. Permeability of the CEC monolayer was assayed by measuring the transendothelial electrical resistance. Results. Exposure of CECs to SNP increased cellular NO levels and simultaneously decreased cell viability (P < .01). Meanwhile, treatment of CECs with propofol at a therapeutic concentration (50 mM) decreased SNP-induced cell death (P < .01). SNP induced DNA fragmentation and cell apoptosis, but propofol decreased the cell injury (P < .01). Sequentially, propofol decreased SNP-enhanced Bak levels and translocation from the cytoplasm to mitochondria (P < .05). Exposure of CECs to propofol attenuated GSNO-induced cell death, apoptosis, and caspase-3 activation (P < .01). Additionally, propofol protected CECs against SNP-induced disruption of the CEC monolayer (P < .05). Consequently, SNPenhanced cascade activation of caspases-9 and -3 was decreased by propofol (P < .01). Conclusion. This study suggested that propofol at a therapeutic concentration can protect against nitrosative stress-induced apoptosis of CECs due to downregulation of the intrinsic Bak-mitochondrioncytochrome c-caspase protease pathway. (Surgery 2013;154:58-68.) From the Graduate Institute of Medical Sciences,a College of Medicine, the Center of Excellence for Cancer Research, Taipei Medical University, the Brain Disease Research Center,b Taipei Medical University-Wan Fang Medical Center, Taipei Medical University, the Anesthetics Toxicology Research Center,c Taipei Medical University Hospital, and the Department of Anesthesiology,d Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Supported by grants from Shin Kong Wu Ho-Su Memorial Hospital (SKH-TMU-99-07; SKH-8302-100-DR-15), the Department of Health (DOH101-TD-C-111-008), Wan-Fang Hospital (100-wf-eva-05), and National Science Council (NSC100-2314B-038-010-MY3; NSC101-2314-B-038-008-MY3; NSC101-2314-B038-005), Taipei, Taiwan. Accepted for publication February 5, 2013. Reprint requests: Gong-Jhe Wu, MD, PhD, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan. E-mail:
[email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2013.02.003
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PROPOFOL (2,6-diisopropylphenol) is an intravenous anaesthetic agent used widely for the induction and maintenance of anaesthesia.1 Clinically, propofol has the benefits of rapid onset, short-action duration, and speedy elimination.2 Propofol is also an effective drug for sedation of patients in the intensive care unit (ICU).3,4 For example, propofol sedation is helpful in young children undergoing precise radiation therapy of brain tumors.5 In addition, propofol is structurally similar to a-tocopherol, so it has antioxidant characteristics.6 Our previous studies showed that propofol can suppress chemotaxis, phagocytosis, and the oxidant ability of macrophages by
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Fig 1. Effects of sodium nitroprusside (SNP) and propofol (PPF) on nitrite production, cell morphologies, and cell viability. Mouse cerebrovascular endothelial cells (CECs) were exposed to 0.1, 0.25, 0.5, 1, or 2 mM SNP for 24 h or to 1 mM SNP for 1, 6, or 24 h. Amounts of nitrite (A) and cell viability (B, C) were analyzed. Mouse CECs were treated with 1, 25, 50, of 100 mM PPF (D) or a combination of 1, 25, or 50 mM PPF and 1 mM SNP (E) for 24 h, and cell viability was determined. After exposure to 1 mM SNP, 50 mM PPF, and a combination of PPF and SNP, cell morphology (F) was assayed. The representative morphologies from a single experiment are shown. Each value represents the mean ± SD for n = 18. Symbols * and # indicate that values significantly differed from the respective control and SNP-treated groups, P < .05.
decreasing mitochondrial activities.7 Moreover, in Gram-positive and -negative bacterium-induced infections, propofol inhibits tumor necrosis factor-a, interleukin-1b, and inducible NO synthase gene expression through downregulating signal-transducing effects mediated by toll-like receptor 2 and 4.8-11 Bacterium-induced infections and subsequent oxidative stress may put ICU patients at high risk for morbidity and mortality. Along with its sedative effects, propofol possesses anti-inflammatory and antioxidative features. Thus, propofol can be chosen beneficially as a
sedative drug for ICU patients with critical illnesses, such as brain tumors and traumatic brain injury. Maintenance of cerebral vessel function is crucial for recuperation by ICU patients with a brain neoplasm or injury. In particular, the blood-brain barrier (BBB) conserves the homeostasis of the cerebral microenvironment.12,13 Disruption of the BBB can lead to worsening of brain diseases.14 Structurally, cerebrovascular endothelial cells (CECs) are fundamental constituents of the BBB.15 CECs form complex tight junctions to drive
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most molecular traffic direction across the BBB.12,15 A variety of intrinsic and extrinsic factors, including infection and oxidative stress, may affect the physiology and pathophysiology of CECs.16,17 De Vries et al18 reported that endotoxin lipopolysaccharide can injure bovine CEC layers. Exposure of human CECs to glutamate decreased the barrier integrity, eventually leading to a breakdown of the BBB.19 Data from previous studies revealed that CEC dysfunction influences the permeability of the BBB, leading to brain damage.12,20 Thus, developing a drug that can protect CECs from such injury is important for the therapeutic treatment of brain diseases. Oxidative stress is one of the critical factors causing CEC insults. Our previous study showed that oxidized low-density lipoprotein can increase cellular oxidative stress and induce CEC apoptosis via a mitochondrion-dependent mechanism.16 Apoptosis can be regulated through both intrinsic and extrinsic pathways.21 Activation of intrinsic mitochondrion-dependent mechanisms involves a series of signal-transducing events, including translocation of a proapoptotic Bcl2-antagonistkiller (Bak) protein from the cytoplasm to the mitochondria, depolarization of the mitochondrial membrane potential, and subsequent release of cytochrome c from mitochondria to the cytoplasm.22,23 After being released into the cytoplasm, cytochrome c can successively activate caspases-9, -3, and -6, which then causes cleavage of key cellular proteins and accordingly damages genomic DNA and induces cell apoptosis.17,24 NO, a reactive oxygen species, can elicit nitrosative stress and induces CEC apoptosis and BBB breakage.25-27 Our previous studies showed that propofol at clinically relevant concentrations can protect macrophages from NO-induced cell insults.28,29 The aim of this study was to evaluate whether propofol can protect the BBB against nitrosative stress-induced damage by decreasing insults to CECs and to determine its possible mechanisms. MATERIALS AND METHODS Isolation of mouse CECs and drug treatment. CECs were prepared from cerebral capillaries of ICR mice as described previously.27 All procedures were approved by the Institutional Animal Care and
=Fig 2.
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Use Committee of Taipei Medical University, Taipei, Taiwan. Mouse CECs were immunocytochemically verified and seeded as described previously.16 Sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) were purchased from Sigma (St. Louis, MO). Propofol was obtained from Aldrich (Milwaukee, WI) and dissolved freshly in dimethyl sulfoxide. Concentrations of propofol used in this study were #50 mM, which are within a range of therapeutic clinical plasma concentrations.30 Assays of cellular NO and cytotoxicity. Amounts of SNP- or GSNO-released NO in mouse CECs were measured as described previously.31 After reaction, a colorimetric azo product was quantified using a photometer (Anthos Labtec Instruments, Wales, Austria). Cell morphology was observed and photographed using a reverse-phasemicroscope, and cell viability was assayed using a colorimetric method to measure spectrophotometrically the absorbance of the blue formazan products at an optical density of 550 nm (OD550).26 Quantification of DNA fragmentation and apoptotic cells. Damage by SNP of the genomic DNA of mouse CECs was determined by analyzing BrdU-labeled histone-associated DNA fragments in cell lysates following the instructions of an ELISA kit (Boehringer Mannheim, Indianapolis, IN).32 Apoptotic cells were quantified following a previous method.33 After drug treatment, mouse CECs were harvested and fixed in cold 80% ethanol. The fixed cells were stained with propidium iodide to detect DNA fragments in nuclei and analyzed using a flow cytometer (FACScan; Becton Dickinson, San Jose, CA). Immunodetection of cellular Bak, cytochrome c, and b-actin. Amounts of Bak and cytochrome c in mouse CECs were immunodetected as described previously.17 The antibodies used for detection of Bak and cytochrome c were a polyclonal antibody against human Bak (Millipore, Temecula, CA) and a monoclonal antibody against pigeon cytochrome c (BioSource, Camarillo, CA). Levels of b-actin were used as the internal control. The intensities of these protein bands were quantified using an UVIDOCMW version 99.03 digital imaging system (UVtec, Cambridge, UK). Confocal analysis of Bak translocation. Bak’s translocation from the cytoplasm to mitochondria
Protection by propofol (PPF) against sodium nitroprusside (SNP)-induced DNA fragmentation and cell apoptosis. Mouse cerebrovascular endothelial cells (CECs) were exposed to 1 mM SNP, 50 mM PPF, or a combination of PPF and SNP. DNA fragmentation (A) and apoptotic cells (B, C) were determined. The representative images of cell cycle analyses from a single experiment are shown. Each value represents the mean ± SD for n $ 12. Symbols * and # indicate that values significantly differed from the respective control and SNP-treated groups, P < .05.
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was assayed by measuring the transendothelial electrical resistance (TEER) using an EVOM resistance meter (World Precision Instruments, Sarasota, FL), as described previously.13 Briefly, mouse CECs were cultured in type-IV collagen-coated transwells (0.4 mm pore size; Corning Costar, Cambridge, MA) at a density of 2 3 105/cm2. The transwells were inserted into 12-well plates containing the medium. The resistance of the CEC monolayer was monitored until stable resistances occurred (>60 X cm2), then cells were treated with resveratrol and oxLDL. After drug treatment, the TEER was measured. Statistical analysis. The statistical significance of differences among control, propofol-, SNP-, and propofol + SNP-treated CECs was evaluated using 1-way ANOVA with the Duncan multiple-range post hoc test (SPSS 18.0 base, IBM Corporation, Endicott, NY), and differences were considered statistically significant at P values of
