Propofol Protects Against High Glucose–Induced Endothelial Apoptosis and Dysfunction in Human Umbilical Vein Endothelial Cells Minmin Zhu, MD, Meilin Wen, MD, Xia Sun, MD, Wankun Chen, MD, Jiawei Chen, MD, PhD, and Changhong Miao, MD, PhD BACKGROUND: Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis and dysfunction. Propofol is a widely used IV anesthetic drug in clinical settings. In the present study, we examined whether and how propofol reduced high glucose–induced endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured with different concentrations (5, 10, 15, and 25 mM) of glucose for different times (4, 8, 12, and 24 hours). To study the effect of propofol, cells were incubated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for 2 hours. In parallel experiments, cells were incubated in 5 mM glucose as control. Nitric oxide (NO) production was measured with a nitrate reductase assay. Cell viability was determined with a Cell Counting Kit-8. Protein expression of active caspase 3, cytochrome c, endothelial NO synthase (eNOS), p-eNOSThr495, p66Shc, protein kinase C βII (PKCβII), and p-PKCβII-Ser660 was measured by Western blot analysis. Accumulation of superoxide anion (O2˙−) was measured with the reduction of ferricytochrome c. Cell apoptosis was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. RESULTS: Compared with control, high glucose decreased NO production (P < 0.0001) and reduced cells viability (P < 0.0001) in HUVECs. Compared with high glucose treatment, pretreatment of cells with propofol (5 μM, 2 hours) reduced high glucose–induced inhibitory p-eNOS-Thr495 phosphorylation (P < 0.0001), increasing NO production (P = 0.0007), decreased high glucose–induced p66Shc expression (P < 0.0001) and p66Shc mitochondrial translocation (P < 0.0001), O2˙− accumulation (P < 0.0001), mitochondrial cytochrome c release (P < 0.0001), active caspase 3 expression (P < 0.0001), and enhancing endothelial viability (P < 0.0001). Furthermore, propofol inhibited high glucose–induced PKCβII expression (P = 0.0002) and p-PKCβII-Ser660 phosphorylation (P < 0.0001). Moreover, the observed protective effect of propofol was quite similar to that of PKCβII inhibitor. CONCLUSIONS: Propofol, by a mechanism of decreasing high glucose–induced PKCβII expression and p-PKCβII-Ser660 phosphorylation, inhibits high glucose–induced p66Shc mitochondrial translocation, therefore protecting HUVECs from high glucose–induced endothelial dysfunction and apoptosis. (Anesth Analg 2015;120:781–9)
P
erioperative hyperglycemia is a common clinical metabolic disorder in nondiabetics1 as well as diabetics2 and has been commonly triggered by physiological stress and excessive glucose transfusion. Hyperglycemia could induce endothelial injury3 and thus has been considered an independent risk factor for augmented perioperative morbidity and mortality.4–7 Reactive oxygen species (ROS) is critically involved in hyperglycemia-induced endothelial injury.8 Mitochondrion is the major site of intracellular biological oxidation and is the most important source of ROS accumulation. The p66Shc adaptor protein functions as a redox enzyme implicated in mitochondrial ROS From the Department of Anaesthesiology, Fudan University Shanghai Cancer Center, and Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China. Accepted for publication November 13, 2014. Funding: None. The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Changhong Miao, MD, PhD, Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, No. 270 DongAn Rd., Shanghai 200032, People’s Republic of China. Address e-mail to [email protected]. Copyright © 2015 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000616
April 2015 • Volume 120 • Number 4
generation and translation of oxidative signals into apoptosis.9 Studies have demonstrated that high glucose could activate protein kinase C βII (PKCβII) and thus facilitate p66Shc mitochondrial translocation, cytochrome c oxidation, and subsequently ROS accumulation.8 ROS accumulation increases mitochondrial permeability, resulting in the release of cytochrome c. Once cytochrome c is released in the cytosol, it triggers the intracellular apoptosis pathway by activating caspase 3, the pivotal apoptosis execution enzyme.10,11 High glucose could also lead to endothelial dysfunction via decreasing nitric oxide (NO) production and increasing ROS accumulation as well.12 A previous study has indicated that high glucose–induced PKCβII activation up-regulated the phosphorylation of endothelial NO synthase (eNOS)-Thr495, thus decreasing NO production.8 High glucose–induced endothelial cell dysfunction and apoptosis (Fig. 1) result in endothelial injury, which may eventually lead to dysfunction of the microvascular environment in multiple organs that cause complications perioperatively. Propofol (2, 6-diisopropylphenol) is a widely used IV anesthetic drug. In our previous studies, propofol was found to improve high glucose–induced endothelial dysfunction13 and inflammation14 and thus attenuating high glucose– induced endothelial injury. Other studies have supportive www.anesthesia-analgesia.org
781
Propofol Protects Against Endothelial Apoptosis and Dysfunction
Figure 1. High glucose activates protein kinase C βII (PKCβII), inducing p66Shc expression and p66Shc mitochondrial translocation, resulting in reactive oxygen species (ROS) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and endothelial cell apoptosis; up-regulating inhibitory endothelial nitric oxide synthasethreonine495 (p-eNOS-Thr495) phosphorylation; and decreasing nitric oxide (NO) production, leading to endothelial dysfunction.
results indicating that propofol could protect endothelial cells from apoptosis in many other situations.15,16 However, the mechanisms by which propofol protects against hyperglycemia-induced endothelial apoptosis is still inconclusive. Our previous study has indicated that propofol could protect against hyperglycemia-induced endothelial dysfunction via recoupling eNOS and up-regulation of p-eNOS-Ser1177 phosphorylation in an hour.13 Whether other protective mechanisms are also involved still needs to be clarified. Human umbilical vein endothelial cells (HUVECs) are widely used to study high glucose–induced endothelial injury in in vitro settings.8 In the present study, we examined whether and how propofol protects HUVECs against high glucose– induced endothelial apoptosis and dysfunction.
METHODS Cell Culture and Reagents
HUVECs (Clonetics; Lonza, Basel, Switzerland) were cultured in Dulbecco Modified Eagle Medium with 5 mM glucose and 10% fetal bovine serum in an incubator containing 5% CO2 at 37°C. Cells were subcultured when reaching 90% confluence. The fourth passage of HUVECs was used in the present study. Propofol (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (Sigma). The final concentration of dimethyl sulfoxide was adjusted to 0.01% for each medium to prevent any possible nonspecific effects. 4, 5-dianilinophthalimide (CGP53353) is a highly selective PKCβII inhibitor. It has been widely used in many studies of PKCβII-involved signal pathways.8 In the present study, incubation of CGP53353 at 10 μM for 2 hours was used to selectively inhibit PKCβII activity.
Study Design
HUVECs were cultured in Dulbecco Modified Eagle Medium with different concentrations (5, 10, 15, and 25 mM) of glucose for different durations (4, 8, 12, and 24 hours). By measuring NO production and endothelial cell viability, we determined the appropriate glucose treatment condition with maximal effect on NO reduction and endothelial cell viability
782 www.anesthesia-analgesia.org
inhibition. During general anesthesia, plasma concentrations of propofol range from 5 to 50 μM.17 HUVECs were therefore incubated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for 2 hours. The optimal concentration of propofol with significant effects on 15 mM glucose–mediated NO production and endothelial cell viability was determined. These treatment conditions were used in subsequent studies in which HUVECs were cultured and divided into 4 groups to examine the underlying signaling pathways. Group 1: HUVECs were cultured in 5 mM glucose as control; group 2: HUVECs were cultured in 5 mM glucose and coincubated with 5 μM propofol for the last 2 hours; group 3: HUVECs were treated with 15 mM glucose for 12 hours; and group 4: HUVECs were treated with 15 mM glucose for 12 hours and coincubated with 5 μM propofol for the last 2 hours.
NO Production Assay
NO production was detected by a nitrate reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China). In brief, cell culture medium was incubated with nitrate reductase, reduced form of nicotinamide adenine denucleotide phosphate, and flavin adenine dinucleotideNa2 and kept at 37°C for 60 minutes. The supernatant was collected after the mixtures were centrifuged at 3500 rpm for 10 minutes. Then, chromogenic agent (sulfanilic acid and N-1-naphthyl-ethylenediamine dihydrochloride) was added in and results were spectrophotometrically determined at 550 nM. Protein concentration was determined by bicinchoninic acid assay. NO production was normalized to the protein concentration of the cells.
Analysis for Cell Viability
Cell suspensions (3000 cells/100 μL) were added to each well in the 96-multiwell culture plate and incubated at 37°C. After corresponding treatments, 10 μL Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Shanghai, China) was then added to each well, and the cells were further incubated for 2 hours. Finally, the optical density at 450 nm was measured using an immunoplate reader. The cell viability curve was determined by calculating the mean value and SD of the optical density for every 6 wells.
Western Blot Analysis
Whole-cell extracts were prepared using cell lysis buffer (Cell Signaling Technology, Danvers, MA), whereas mitochondrial extracts and cytosol extracts were prepared with a mitochondrial extract kit (Shanghai Shengong Bioengineering Institute, Shanghai City, P. R. China). Equal amounts (30 μg) of protein extracted from different groups of HUVECs were separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thereafter transferred to polyvinylidene difluoride membranes. After being blocked in 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies were monoclonal antibody against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome c oxidase IV (COX IV; Santa Cruz Biotechnology), eNOS (Santa Cruz Biotechnology), p-eNOS-Thr495 (Santa Cruz Biotechnology), caspase 3 (Epitomics), PKCβII (Epitomics, Burlingame, CA), p-PKCβII-Ser660 (Abcam, Cambridge,
anesthesia & analgesia
MA), p66Shc (Epitomics), and cytochrome C (Epitomics). Thereafter, the primary antibodies were washed away, and the membranes were incubated with secondary antibodies for 1 hour at room temperature. Subsequently, the membranes were washed 3 times with Tris-buffered saline with Tween and detected by the ECL system (Beyotime Institute of Biotechnology, Shanghai, China). The respective densities of the protein bands were analyzed by Scan-gel-it software (UNSCAN-IT gel 6.0, Silk Scientific Inc., Orem, UT). In the present study, β-actin was used as the loading control in whole-cell extracts and cytosol extracts, whereas COX IV was used as loading control in mitochondrial extracts. Also, the data were interpreted as the ratio of specific protein expression and β-actin expression or COX IV.
Superoxide Anion (O2˙−) Accumulation Assay
Superoxide anion (O2˙−) accumulation was measured by the reduction of ferricytochrome c assay as described previously.3 In brief, cell suspensions (3000 cells/100 μL) were added to each well in the 96-multiwell culture plate and incubated at 37°C. After corresponding treatments, cells were washed and cultured with Krebs-HEPES buffer containing 20 μM ferricytochrome c (Sigma) with or without superoxide dismutase (Sigma). The absorbance was spectrophotometrically read at 550 nm. Reduction of ferricytochrome c with superoxide dismutase was subtracted from the data without superoxide dismutase. Arbitrary unit was used as the unit for absorbance difference.
TUNEL Staining
Cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with a commercially available kit (Shanghai Yusheng Bioengineering Institute, Shanghai, China). In brief, HUVECs, cultured on Chamber Slides (Shanghai Haoran Bioengineering Institute, Shanghai, China), were fixed with 4% paraformaldehyde and permealized with proteinase K. Samples were incubated in equilibration buffer at room temperature for 20 minutes and then cultured with TdT incubation buffer for 1 hour at 37°C in the dark. Nuclei were stained with DAPI (blue). TUNEL-positive nuclei (red) were counted under a fluorescence microscope. The data were expressed as the percentage of the control group.
Statistical Analysis
For the present experiment, the sample size was determined based on an assessment of the magnitude of the protective effect of propofol against high glucose–induced endothelial injury which was observed in our pilot experiments, and we anticipated that statistical significance could be achieved with the sample size of 6 in every experiment. Results were obtained from 6 separately performed experiments and are expressed as mean ± SD. N represents the times of repeated experiments using different cell cultures. Statistical comparison was performed with 1-way analysis of variance followed with Bonferroni-corrected pairwise comparisons. In the present study, there are 21 or 6 pairwise combinations in each experiment, and the calculated P values are therefore reported after being multiplied by either 21 or 6, as appropriate. A post hoc value of P
P
erioperative hyperglycemia is a common clinical metabolic disorder in nondiabetics1 as well as diabetics2 and has been commonly triggered by physiological stress and excessive glucose transfusion. Hyperglycemia could induce endothelial injury3 and thus has been considered an independent risk factor for augmented perioperative morbidity and mortality.4–7 Reactive oxygen species (ROS) is critically involved in hyperglycemia-induced endothelial injury.8 Mitochondrion is the major site of intracellular biological oxidation and is the most important source of ROS accumulation. The p66Shc adaptor protein functions as a redox enzyme implicated in mitochondrial ROS From the Department of Anaesthesiology, Fudan University Shanghai Cancer Center, and Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China. Accepted for publication November 13, 2014. Funding: None. The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Changhong Miao, MD, PhD, Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, No. 270 DongAn Rd., Shanghai 200032, People’s Republic of China. Address e-mail to [email protected]. Copyright © 2015 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000616
April 2015 • Volume 120 • Number 4
generation and translation of oxidative signals into apoptosis.9 Studies have demonstrated that high glucose could activate protein kinase C βII (PKCβII) and thus facilitate p66Shc mitochondrial translocation, cytochrome c oxidation, and subsequently ROS accumulation.8 ROS accumulation increases mitochondrial permeability, resulting in the release of cytochrome c. Once cytochrome c is released in the cytosol, it triggers the intracellular apoptosis pathway by activating caspase 3, the pivotal apoptosis execution enzyme.10,11 High glucose could also lead to endothelial dysfunction via decreasing nitric oxide (NO) production and increasing ROS accumulation as well.12 A previous study has indicated that high glucose–induced PKCβII activation up-regulated the phosphorylation of endothelial NO synthase (eNOS)-Thr495, thus decreasing NO production.8 High glucose–induced endothelial cell dysfunction and apoptosis (Fig. 1) result in endothelial injury, which may eventually lead to dysfunction of the microvascular environment in multiple organs that cause complications perioperatively. Propofol (2, 6-diisopropylphenol) is a widely used IV anesthetic drug. In our previous studies, propofol was found to improve high glucose–induced endothelial dysfunction13 and inflammation14 and thus attenuating high glucose– induced endothelial injury. Other studies have supportive www.anesthesia-analgesia.org
781
Propofol Protects Against Endothelial Apoptosis and Dysfunction
Figure 1. High glucose activates protein kinase C βII (PKCβII), inducing p66Shc expression and p66Shc mitochondrial translocation, resulting in reactive oxygen species (ROS) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and endothelial cell apoptosis; up-regulating inhibitory endothelial nitric oxide synthasethreonine495 (p-eNOS-Thr495) phosphorylation; and decreasing nitric oxide (NO) production, leading to endothelial dysfunction.
results indicating that propofol could protect endothelial cells from apoptosis in many other situations.15,16 However, the mechanisms by which propofol protects against hyperglycemia-induced endothelial apoptosis is still inconclusive. Our previous study has indicated that propofol could protect against hyperglycemia-induced endothelial dysfunction via recoupling eNOS and up-regulation of p-eNOS-Ser1177 phosphorylation in an hour.13 Whether other protective mechanisms are also involved still needs to be clarified. Human umbilical vein endothelial cells (HUVECs) are widely used to study high glucose–induced endothelial injury in in vitro settings.8 In the present study, we examined whether and how propofol protects HUVECs against high glucose– induced endothelial apoptosis and dysfunction.
METHODS Cell Culture and Reagents
HUVECs (Clonetics; Lonza, Basel, Switzerland) were cultured in Dulbecco Modified Eagle Medium with 5 mM glucose and 10% fetal bovine serum in an incubator containing 5% CO2 at 37°C. Cells were subcultured when reaching 90% confluence. The fourth passage of HUVECs was used in the present study. Propofol (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (Sigma). The final concentration of dimethyl sulfoxide was adjusted to 0.01% for each medium to prevent any possible nonspecific effects. 4, 5-dianilinophthalimide (CGP53353) is a highly selective PKCβII inhibitor. It has been widely used in many studies of PKCβII-involved signal pathways.8 In the present study, incubation of CGP53353 at 10 μM for 2 hours was used to selectively inhibit PKCβII activity.
Study Design
HUVECs were cultured in Dulbecco Modified Eagle Medium with different concentrations (5, 10, 15, and 25 mM) of glucose for different durations (4, 8, 12, and 24 hours). By measuring NO production and endothelial cell viability, we determined the appropriate glucose treatment condition with maximal effect on NO reduction and endothelial cell viability
782 www.anesthesia-analgesia.org
inhibition. During general anesthesia, plasma concentrations of propofol range from 5 to 50 μM.17 HUVECs were therefore incubated with different concentrations (0.2, 1, 5, and 25 μM) of propofol for 2 hours. The optimal concentration of propofol with significant effects on 15 mM glucose–mediated NO production and endothelial cell viability was determined. These treatment conditions were used in subsequent studies in which HUVECs were cultured and divided into 4 groups to examine the underlying signaling pathways. Group 1: HUVECs were cultured in 5 mM glucose as control; group 2: HUVECs were cultured in 5 mM glucose and coincubated with 5 μM propofol for the last 2 hours; group 3: HUVECs were treated with 15 mM glucose for 12 hours; and group 4: HUVECs were treated with 15 mM glucose for 12 hours and coincubated with 5 μM propofol for the last 2 hours.
NO Production Assay
NO production was detected by a nitrate reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China). In brief, cell culture medium was incubated with nitrate reductase, reduced form of nicotinamide adenine denucleotide phosphate, and flavin adenine dinucleotideNa2 and kept at 37°C for 60 minutes. The supernatant was collected after the mixtures were centrifuged at 3500 rpm for 10 minutes. Then, chromogenic agent (sulfanilic acid and N-1-naphthyl-ethylenediamine dihydrochloride) was added in and results were spectrophotometrically determined at 550 nM. Protein concentration was determined by bicinchoninic acid assay. NO production was normalized to the protein concentration of the cells.
Analysis for Cell Viability
Cell suspensions (3000 cells/100 μL) were added to each well in the 96-multiwell culture plate and incubated at 37°C. After corresponding treatments, 10 μL Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Shanghai, China) was then added to each well, and the cells were further incubated for 2 hours. Finally, the optical density at 450 nm was measured using an immunoplate reader. The cell viability curve was determined by calculating the mean value and SD of the optical density for every 6 wells.
Western Blot Analysis
Whole-cell extracts were prepared using cell lysis buffer (Cell Signaling Technology, Danvers, MA), whereas mitochondrial extracts and cytosol extracts were prepared with a mitochondrial extract kit (Shanghai Shengong Bioengineering Institute, Shanghai City, P. R. China). Equal amounts (30 μg) of protein extracted from different groups of HUVECs were separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thereafter transferred to polyvinylidene difluoride membranes. After being blocked in 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies were monoclonal antibody against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome c oxidase IV (COX IV; Santa Cruz Biotechnology), eNOS (Santa Cruz Biotechnology), p-eNOS-Thr495 (Santa Cruz Biotechnology), caspase 3 (Epitomics), PKCβII (Epitomics, Burlingame, CA), p-PKCβII-Ser660 (Abcam, Cambridge,
anesthesia & analgesia
MA), p66Shc (Epitomics), and cytochrome C (Epitomics). Thereafter, the primary antibodies were washed away, and the membranes were incubated with secondary antibodies for 1 hour at room temperature. Subsequently, the membranes were washed 3 times with Tris-buffered saline with Tween and detected by the ECL system (Beyotime Institute of Biotechnology, Shanghai, China). The respective densities of the protein bands were analyzed by Scan-gel-it software (UNSCAN-IT gel 6.0, Silk Scientific Inc., Orem, UT). In the present study, β-actin was used as the loading control in whole-cell extracts and cytosol extracts, whereas COX IV was used as loading control in mitochondrial extracts. Also, the data were interpreted as the ratio of specific protein expression and β-actin expression or COX IV.
Superoxide Anion (O2˙−) Accumulation Assay
Superoxide anion (O2˙−) accumulation was measured by the reduction of ferricytochrome c assay as described previously.3 In brief, cell suspensions (3000 cells/100 μL) were added to each well in the 96-multiwell culture plate and incubated at 37°C. After corresponding treatments, cells were washed and cultured with Krebs-HEPES buffer containing 20 μM ferricytochrome c (Sigma) with or without superoxide dismutase (Sigma). The absorbance was spectrophotometrically read at 550 nm. Reduction of ferricytochrome c with superoxide dismutase was subtracted from the data without superoxide dismutase. Arbitrary unit was used as the unit for absorbance difference.
TUNEL Staining
Cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with a commercially available kit (Shanghai Yusheng Bioengineering Institute, Shanghai, China). In brief, HUVECs, cultured on Chamber Slides (Shanghai Haoran Bioengineering Institute, Shanghai, China), were fixed with 4% paraformaldehyde and permealized with proteinase K. Samples were incubated in equilibration buffer at room temperature for 20 minutes and then cultured with TdT incubation buffer for 1 hour at 37°C in the dark. Nuclei were stained with DAPI (blue). TUNEL-positive nuclei (red) were counted under a fluorescence microscope. The data were expressed as the percentage of the control group.
Statistical Analysis
For the present experiment, the sample size was determined based on an assessment of the magnitude of the protective effect of propofol against high glucose–induced endothelial injury which was observed in our pilot experiments, and we anticipated that statistical significance could be achieved with the sample size of 6 in every experiment. Results were obtained from 6 separately performed experiments and are expressed as mean ± SD. N represents the times of repeated experiments using different cell cultures. Statistical comparison was performed with 1-way analysis of variance followed with Bonferroni-corrected pairwise comparisons. In the present study, there are 21 or 6 pairwise combinations in each experiment, and the calculated P values are therefore reported after being multiplied by either 21 or 6, as appropriate. A post hoc value of P
