American Journal of Therapeutics 0, 1–8 (2015)
Beta Blockers Suppress Dextrose-Induced Endoplasmic Reticulum Stress, Oxidative Stress, and Apoptosis in Human Coronary Artery Endothelial Cells Michael J. Haas, PhD, William Kurban, MD, Harshit Shah, MD, Luisa Onstead-Haas, MS, and Arshag D. Mooradian, MD*
Beta blockers are known to have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. To determine whether beta blockers can also prevent dextrose-induced endoplasmic reticulum (ER) stress in addition to their antioxidative effects, human coronary artery endothelial cells and hepatocyte-derived HepG2 cells were treated with 27.5 mM dextrose for 24 hours in the presence of carvedilol (a lipophilic beta blockers with alpha blocking activity), propranolol (a lipophilic nonselective beta blockers), and atenolol (a water-soluble selective beta blockers), and ER stress, oxidative, stress and cell death were measured. ER stress was measured using the placental alkaline phosphatase assay and Western blot analysis of glucose regulated protein 78, c-Jun-N-terminal kinase (JNK), phospho-JNK, eukaryotic initiating factor 2a (eIF2a), and phospho-eIF2a and measurement of X-box binding protein 1 (XBP1) mRNA splicing using reverse transcriptase-polymerase chain reaction. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A] pyrazin-3-one hydrochloride (MCLA) chemiluminescence. Cell viability was measured by propidium iodide staining method. The ER stress, SO production, and cell death induced by 27.5 mM dextrose were inhibited by all 3 beta blockers tested. The antioxidative and ER stress reducing effects of beta blockers were also observed in HepG2 cells. The salutary effects of beta blockers on endothelial cells in reducing both ER stress and oxidative stress may contribute to the cardioprotective effects of these agents. Keywords: beta blockers, endothelial cells, HepG2 cells, endoplasmic reticulum stress, cardiovascular disease
INTRODUCTION Cardiovascular disease is the leading cause of morbidity and mortality in people with diabetes mellitus.1,2 Only few classes of therapeutic agents are found to be effective in reducing cardiovascular event rates. These include antithrombotic agents, angiotensin converting enzyme Department of Medicine, University of Florida College of Medicine, Jacksonville, FL. Supported by Dean’s Fund Research Grant from the University of Florida College of Medicine, Jacksonville, FL. The authors have no conflicts of interest to declare. *Address for correspondence: Department of Medicine, University of Florida College of Medicine, 653-1 West Eighth St, Jacksonville, FL 32209. E-mail: [email protected]
inhibitors, or angiotensin receptor blockers, statins and beta blockers.3 Although the pharmacology of these agents is well characterized, the precise mechanisms underlying their cardioprotective properties are not entirely understood. The beneficial effects of beta blockers are attributed to their antiarrhythmogenic activity and their effectiveness in reducing myocardial oxygen demand through reducing the heart rate and myocardial contractility.4,5 However, known antiarrythmogenic drugs have either failed to improve patient outcomes and in some instances have increased the risk of mortality.6,7 Furthermore, calcium channel blockers that can reduce the heart rate and myocardial contractility have not been consistently associated with survival advantage, although they can be effective agents in controlling angina.8,9 Furthermore, ivabradine, an agent that selectively reduces the heart rate, was recently shown
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to have no benefit in patients with coronary artery disease.10 Thus, it is likely that the known cardioprotective properties of beta blockers are the results of biological changes that extend beyond their known effects on heart rate and myocardial contractility. Previously published studies have suggested that some beta blockers have antioxidative activity that can alter endothelial cell expression of vasoactive molecules and have effects on the proliferation and apoptosis of human coronary artery smooth muscle and endothelial cells.11–14 In the present communication, we report on an additional pleiotropic effect of beta blockers that may contribute to their cardioprotective effects, namely their ability to reduce ER stress.
MATERIALS AND METHODS Materials
pSEAP2-Control, containing a truncated placental alkaline phosphatase gene adjacent to the simian virus 40 early gene promoter, and 24 hours later treated with 5.5 or 27.5 mM dextrose with the carvedilol, propranolol, and atenolol indicated amounts or the solvent dimethyl sulfoxide (DMSO). After 24 hours, secreted alkaline phosphatase (SAP) activity was measured by mixing 25-mL of conditioned medium with 75-mL of dilution buffer, which was incubated at 65°C for 30 minutes in 1.5 mL microcentrifuge tubes. After cooling on ice for 3 minutes, 100 mL of CSPD was added to each tube. The samples were incubated at room temperature for 30 minutes, and SAP activity was measured with a Modulus luminometer (Promega, Madison, WI) and is expressed in relative light units (RLU). As a negative control, SAP activity was measured in cells transfected with pSEAP2-Basic, which contains the placental SAP gene but lacks a eukaryotic promoter to drive expression. The solvent DMSO (diluted 1: 1000) had no effect on SAP activity.
Carvedilol was obtained from Sigma–Aldrich (St. Louis, MO), atenolol was obtained from MP Biomedicals (Santa Ana, CA), and propranolol was obtained from Enzo Life Sciences (Farmingdale, NY). The chemiluminescent alkaline phosphatase substrate 3-(diethylamino)-1-(2,2-dimethyl-3-H-1-benzofuran7-yl)-propan-1-one (CSPD) was purchased from Clontech Laboratories Inc. (Mountain View, CA). Lipofectamine and the superoxide-reactive probe 2-Methyl-6-(4Methoxyphenyl)-3,7-Dihydroimidazo[1,2-A]pyrazin3-one hydrochloride (MCLA) were obtained from Life Technologies (Carlsbad, CA) All other chemicals were obtained from Sigma–Aldrich or Fisher Scientific (Pittsburgh, PA).
Superoxide (SO) generation was measured using MCLA chemiluminescence as previously described.17 HCAEC and HepG2 cells were treated with 5.5 or 27.5 mM dextrose with increasing amounts of carvedilol, atenolol, and propranolol in 6-well plates, and MCLA was added to a final concentration of 1 mmol/L in Hank balanced salt solution containing 1.26 mM CaCl2, 5.37 mM KCl, 0.44 mM KH2PO4, 0.49 mM MgCl2$6H2O, 0.41 mM MgSO4$7H2O, 136.7 mM NaCl, 4.2 mM NaHCO3, 0.34 mM Na2HPO4, and 5.5 mM D-glucose. Superoxide-induced chemiluminescence was measured with a luminometer and is expressed in RLU.
Cell culture
Western blotting
Human coronary artery endothelial cells (HCAEC) purchased from Life Technologies were maintained in phenol red-free Medium 200 (Life Technologies) containing 1% low serum growth supplement. The hepatoblastoma cell line HepG2 was purchased from American Type Culture Collection and were maintained in BioWhittaker Dulbecco modified essential medium (Fisher Scientific) containing 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 units per milliliter of penicillin. The HCAEC and HepG2 cells were maintained in a humidified incubator at 37°C and 5% CO2.
HCAEC were treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, and propranolol and after 24 hours later, protein extracts were prepared by suspending the cells in 200 mL of lysis buffer (12.5% glycerol, 40 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 8.0), 5% 2-mercaptoethanol, and 1% sodium dodecylsulfate). Protein content was measured using the Bradford assay,18 and 50 mg of protein extract was fractionated by electrophoresis on a 10%-sodium dodecylsulfate polyacrylamide gel.19 After transfer to nitrocellulose,20 the membrane was blocked with 10% newborn calf serum in Tris-buffered saline/Tween 20 (TBST; 50 mM Tris-Cl, 150 mM NaCl, and 0.5% Tween 20, pH 7.4) and incubated overnight with antibodies to glucose regulated protein 78 (GRP78), c-Jun-N-terminal kinase (JNK), phospho-JNK (all from Cell Signaling Technology, Danvers, MA) (all diluted 1:750), or eukaryotic
ER stress measurement ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay15,16 (Great EscAPe; Clontech Laboratories Inc, Mountain View, CA). Briefly, HCAEC and HepG2 cells at 70%–80% confluence in 6-well plates were transfected with the plasmid American Journal of Therapeutics (2015) 0(0)
Superoxide generation
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Beta Blockers Suppress ER Stress
initiating factor 2a (eIF2a), and phospho-eIF2a (diluted 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Southern Biotech., Birmingham, AL) (diluted 1:5000), and binding was detected by enhanced chemiluminescence with reagents from Pierce Biotechnology (Rockford, IL). X-box binding protein 1 (XBP1) mRNA splicing Total RNA was isolated from endothelial cells treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, and propranolol for 24 hours. One microgram of RNA was reverse transcribed, and 100 ng of complementary DNA was used in each polymerase chain reaction with the following primers. XBP1 forward, 59-TTA CGA GAG AAA ACT CAT GGC C-39; XBP1 reverse, 59-GGG TCC AAG TTG TCC AGA ATG C-39. After polymerase chain reaction, the samples were fractionated by electrophoresis on a 3% agarose gel in Tris-borate-EDTA [TBE; 89 mM Tris-Cl, 89 mM boric acid, 2 mM ethylenediamine tetraacetic acid (EDTA)]. The gel was stained with ethidium bromide and photographed under ultraviolet light. Measurement of endothelial cell viability HCAEC were cultured in 6-well plates and treated with 5.5 or 27.5 mM dextrose with and without 10 mM carvedilol, propranolol, or atenolol. After 24 and 48 hours, propidium iodide (PI) was added at a final concentration of 10 mg/mL, and the cultures were incubated with the cells for 5 minutes as previously described.21 The cells were visualized under ultraviolet light (340–380 nM), and the number of PI-positive cells was counted in 4 fields, over 150 cells per field. Statistical analysis All results are expressed as mean 6 SD. Analysis of variance followed by the Newman–Keuls procedure for subgroup analysis was performed using the statistical package Statistica for Windows (Statsoft Inc, Tulsa, OK). Significance was defined as a 2-tailed P , 0.05. The half maximal effective concentration (EC50) for carvedilol, atenolol, and propranolol effects on SO generation, and ER stress in HCAEC and HepG2 cells was obtained from nonlinear regression of the dose response curves using GraphPad Prism 5 (La Jolla, CA).
RESULTS Effect of beta blockers on dextrose-induced ER stress in HCAEC The SAP activity decreased from 248,263 6 20,483 RLU in control cells treated with 5.5 mM dextrose www.americantherapeutics.com
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to 164,775 6 13,629 RLU in cells treated with 27.5 mM dextrose (P , 0.001), indicating increased ER stress (Figure 1A). Addition of carvedilol increased SAP activity to 160,517 6 19,544, 201,609 6 18,164, 261,546 6 16,189 RLU in cells treated with 0.1, 1.0, and 10 mM carvedilol (nonsignificant [NS], P , 0.05, and P , 0.001, respectively) (Figure 1A). Treatment of cells with 0.1, 1.0, and 10 mM atenolol increased SAP activity from a baseline of 177,627 6 110,772 RLU to 192,670 6 13,154, 213,978 6 9826, and 266,393 6 11,187 RLU (NS, P , 0.01, and P , 0.001, respectively) (Figure 1B), and treatment of cells with 0.1, 1.0, and 10 mM propranolol increased SAP activity from a baseline of 134,424 6 12,762 RLU to 133,003 6 7353, 159,604 6 16,703, and 177,214 6 1152 RLU (NS, P , 0.05, and P , 0.01, respectively) (Figure 1C). Addition of carvedilol, atenolol, or propranolol (Figures 2A–C) had no effect on SAP activity in HCAEC treated with 5.5 mM dextrose (all NS). These results indicate that all 3 beta blockers inhibit dextrose-induced ER stress in HCAEC with an EC50 of 1.5, 1.6, and 1.1 mM for carvedilol, atenolol, and propranolol, respectively (Table 1). Effect of beta blockers on ER stress markers Treatment of HCAEC with 27.5 mM dextrose increased GRP 78 expression, eIF2a phosphorylation, and JNK phosphorylation relative to cells treated with 5.5 mM dextrose (Figure 2A). In contrast, when HCAEC were treated with 10 mM carvedilol, 10 mM atenolol, and 10 mM propranolol, and 27.5 mM dextrose, GRP 78 expression was similar to HCAEC treated with 5.5 mM dextrose (Figure 2A). Likewise, eIF2a and JNK phosphorylation in carvedilol-, atenolol-, and propranolol-treated cells exposed to 27.5 mM dextrose was similar to eIF2a and JNK phosphorylation in HCAEC exposed to 5.5 mM dextrose (Figure 2A). Splicing of the XBP1 mRNA is also associated with induction of ER stress.16 Treatment of HCAEC with 27.5 mM dextrose induced XBP1 mRNA splicing (Figure 2B, lanes 2 and 3). Addition of carvedilol (10 mM), atenolol (10 mM), or propranolol (10 mM) inhibited XBP1 mRNA splicing (Figure 2B, lanes 4–6). These results further confirm that all 3 beta blockers examined inhibit ER stress in HCAEC. Effect of beta blockers on SO generation in HCAEC Treating HCAEC with high ambient dextrose concentration increased SO generation from a baseline of 5102 6 197 RLU to 6376 6 146 RLU in cells exposed to 27.5 mM dextrose (P , 0.009) (Figure 3A). Addition of 0.1, 1.0, and 10 mM carvedilol decreased SO American Journal of Therapeutics (2015) 0(0)
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FIGURE 1. The effect of 0, 0.1, 1.0, and 10 mM carvedilol, atenolol, and propranolol on dextrose-induced ER stress in HCAEC treated with 5.5 or 27.5 mM dextrose. ER stress (inverse of SAP activity) was measured 24 hours after treating the cells. (A) Carvedilol (CV). N 5 6; *P , 0.001, relative to cells treated with 5.5 mM dextrose; †P , 0.05 and P , 0.001, relative to cells treated with 27.5 mM dextrose. (B) Atenolol (Aten.). N 5 6; *P , 0.003, relative to cells treated with 5.5 mM dextrose; †P , 0.01 and P , 0.001, relative to cells treated with 27.5 mM dextrose. (C) Propranolol (Prop.). N 5 6; *P , 0.006, relative to cells treated with 5.5 mM dextrose; †P , 0.05 and P , 0.01, relative to cells treated with 27.5 mM dextrose. †P-values shown are for 0.1, 1.0, and 10 mM concentration of the agents used, respectively.
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FIGURE 2. The effect of carvedilol, atenolol, and propranolol on ER stress markers in human coronary artery endothelial cells. (A) Cells were treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, or propranolol for 24 hours. GRP 78, phospho-eIF2a, eIF2a, phospho-JNK, and JNK were measured by Western blotting. (B) XBP1 mRNA splicing was examined by reverse-transcription polymerase chain reaction. Treatment with 27.5 mM dextrose increased GRP 78 levels and phospho-eIF2a and phospho-JNK levels (A) and splicing of the XBP1 mRNA (B). Treatment with carvedilol, atenolol, and propranolol decreased GRP 78, phospho-eIF2a, phospho-JNK levels (A) and decreased the amount of the XBP1 spliced mRNA levels (B).
generation to 5558 6 186, 5055 6 180, and 5212 6 128 RLU, respectively (P , 0.004, 0.006, and 0.0005, respectively) (Figure 3A). Treating cells with 0.1, 1.0, and 10 mM atenolol decreased SO generation in HCAEC exposed to 27.5 mM dextrose from a baseline of 6335 6 633 RLU to 6244 6 765, 5076 6 405, and 3599 6 492 RLU, respectively (NS, P , 0.04 and 0.004, respectively) (Figure 3B), and treating cells with 0.1, 1.0, and 10 mM propranolol reduced SO generation www.americantherapeutics.com
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Table 1. Effect of carvedilol, atenolol, and propranolol on SO generation and ER stress in HCAEC and hepatomaderived (HepG2) cells. Carvedilol Atenolol Propranolol EC50, mM EC50, mM EC50, mM HCAEC SO generation ER stress HepG2 SO generation ER stress
0.6 1.5
1.3 1.6
0.8 1.1
0.1 2.3
1.4 1.8
2.2 2.0
EC50 values were calculated from the data in Figures 1, 2 and dose response curves performed with HepG2 cells.
in HCAEC exposed to 27.5 mM dextrose from a baseline of 11,805 6 1232 RLU to 11,799 6 964, 9104 6 1436, and 6612 6 1325 RLU, respectively (NS, NS, and P , 0.004, respectively) (Figure 3C). Addition of carvedilol, atenolol, or propranolol (Figures 3A–C) had no effect on ROS generation in HCAEC treated with 5.5 mM dextrose (all NS). These results indicate that all 3 beta blockers inhibit dextrose-induced SO generation in HCAEC with an EC50 of 0.6, 1.3, and 0.8 mM for carvedilol, atenolol, and propranolol, respectively (Table 1). Effect of beta blockers on endothelial cell viability Long-term exposure of HCAEC to hyperglycemic conditions leads to enhanced cell death.16,17 To determine whether carvedilol, atenolol, and propranolol can prevent dextrose-induced endothelial cell death, HCAEC were treated with 5.5 and 27.5 mM dextrose for 24 (Figure 4A) and 48 hours (Figure 4B) in the presence or absence of 10 mM carvedilol, 10 mM atenolol, or 10 mM propranolol. Addition of carvedilol, atenolol, and propranolol decreased the number of PI staining cells at both 24 and 48 hours. Effect of beta blockers on dextrose-induced ER stress and SO generation in HepG2 cells To determine whether carvedilol, atenolol, and propranolol inhibit ER stress and SO generation in nonendothelial cells, HepG2 cells were treated with 5.5 and 27.5 mM dextrose, and ER stress (Figure 5A) and SO generation (Figure 5B) were measured as described above. High concentrations of dextrose reduced SAP expression from 108,893 6 6956 RLU to 82,502 6 2837 RLU (P , 0.004). Addition of 10 mM carvedilol, 10 mM atenolol, and 10 mM propranolol increased SAP expression to 10,697 6 4796, www.americantherapeutics.com
FIGURE 3. The effect of 0, 0.1, 1.0, and 10 mM carvedilol, atenolol, and propranolol on dextrose-induced superoxide (SO) generation in HCAEC treated with 5.5 or 27.5 mM dextrose. The SO generation was measured 24 hours after treatment of cells by MCLA chemiluminescence. (A) Carvedilol (CV). N 5 6; *P , 0.009, relative to cells treated with 5.5 mM dextrose; †P , 0.004, P , 0.006, and P , 0.0005, relative to cells treated with 27.5 mM dextrose. (B) Atenolol (Aten.). N 5 6; *P , 0.004, relative to cells treated with 5.5 mM dextrose; †P , 0.04 and P , 0.004, relative to cells treated with 27.5 mM dextrose. (C) Propranolol (Prop.). N 5 6; *P , 0.002, relative to cells treated with 5.5 mM dextrose; †P , 0.004, relative to cells treated with 27.5 mM dextrose. †P values shown are for 0.1, 1.0, and 10 mM concentration of the agents used, respectively.
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Haas et al
FIGURE 4. The effect of carvedilol, atenolol, and propranolol on dextrose-induced human coronary artery endothelial cell viability. Cells were treated with either 5.5 or 27.5 mM dextrose for 24 hours (A) or 48 hours (B) in the presence of 10 mM carvedilol (CV), 10 mM atenolol (Aten.), or 10 mM propranolol (Prop.). Cell viability was measured by staining with PI, and dying cells were counted under fluorescent illumination. Treatment of cells with 27.5 mM dextrose increased the number of PI-positive cells at both 24 and 48 hours. Addition of carvedilol, atenolol, and propranolol decreased the number of PI-positive cells at both 24 and 48 hours. (A) *P , 0.07, relative to cells treated with 5.5 mM dextrose; †P , 0.05, P , 0.06, and P , 0.07, relative to cells treated with 27.5 mM dextrose. (B) *P , 0.01, relative to cells treated with 5.5 mM dextrose; †P , 0.008, P , 0.005, and P , 0.09, relative to cells treated with 27.5 mM dextrose. †P values shown are for carvedilol, atenolol, and propranolol, respectively.
FIGURE 5. The effect of carvedilol, atenolol, and propranolol on superoxide (SO) generation and ER stress in HepG2 cells. (A) HepG2 cells were treated with 5.5 and 27.5 mM dextrose with or without 10 mM carvedilol (CV), 10 mM atenolol (Aten.), or 10 mM propranolol (Prop.), and SO generation was measured. (B) HepG2 cells were transfected with pSEAP-Control and 24 hours later treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, 10 mM atenolol, or 10 mM propranolol. After 24 hours, SAP activity was measured. Carvedilol, atenolol, and propranolol decreased SO production and ER stress in HepG2 cells treated with 27.5 mM dextrose. (A) N 5 6; *P , 0.0004, relative to cells treated with 5.5 mM dextrose; †P , 0.002, P , 0.008, P , 0.005, relative to cells treated with 27.5 mM dextrose. (B) N 5 6; *P , 0.002, relative to cells treated with 5.5 mM dextrose; †P , 0.0005, P , 0.001, P , 0.002, relative to cells treated with 27.5 mM dextrose. †P values shown are for carvedilol, atenolol, and propranolol, respectively.
110,938 6 4674, and 107,649 6 7210 RLU, respectively (P , 0.002, 0.0008, and 0.005, respectively). High ambient concentrations of dextrose induced SO generation in HepG2 cells (Figure 5B) from 3664 6 254 RLU to 4926 6 175 RLU (P , 0.002). Addition of 10 mM carvedilol, 10 mM atenolol, and 10 mM
propranolol decreased SO generation to 3638 6 117, 3788 6 160, and 3747 6 214 RLU, respectively (P , 0.0005, 0.001, and 0.002, respectively). In these cells, the EC50 for carvedilol, atenolol, and propranolol in reducing SO generation was 0.1, 1.4, and 2.2 mM, respectively, and for ER stress reduction, the EC50s were
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Beta Blockers Suppress ER Stress
2.3, 1.8, and 2.0 mM, respectively (Table 1). These results indicate that the 3 beta blockers tested inhibit ER stress and oxidative stress in HepG2 cells and in HCAEC.
DISCUSSION Human endothelial cells in culture contain ample betaadrenergic receptors, mostly the beta 2-subtype.22 These receptors are implicated in the regulation of renin–angiotensin system of the endothelial cells23 and in the production of other vasoactive peptides such as endothelin 1.11,14 In addition, previous studies have shown that beta-adrenergic receptor blockers, notably carvedilol, have a potent antioxidant activity11,12 and may also have favorable effects on cell survival.11,13 This study confirms the previously reported antioxidative properties of beta blockers and more importantly demonstrates for the first time that beta blockers as a class may be effective in reducing the ER and oxidative stress simultaneously. The 3 agents tested were effective in both endothelial cells and the HepG2 cells, although the differences in the potency of these 3 agents were less apparent in the HepG2 cells. Analysis of concentration–response curves reveals that in endothelial cells, carvedilol is the most potent of the 3 as to its antioxidative potential with an EC50 that as 25%–50% lower than that of propranolol and atenolol, respectively, whereas in HepG2 cells, the EC50 of carvedilol for inhibition of SO generation was 10- to 20-fold lower than of atenolol and propranolol (Table 1). In contrast, the potency of these 3 beta blockers in reducing ER stress was comparable, and overall, they appeared to be more potent ER stress suppressors in endothelial cells than in HepG2 cells (Table 1). Although there may be a link between oxidative stress and ER stress, the 2 pathways can be dissociated and independent.24,25 The proximal signals for these 2 stresses are generated by different intermediaries of glucose metabolism,16 and whereas antioxidant vitamins are effective in reducing oxidative stress that do not attenuate ER stress.17 Thus, there seems to be limited classes of agents that are effective in reducing oxidative stress and ER stress simultaneously. In this category of dual stress reducers, statins have been shown to be effective in endothelial cells and in HepG2 cells.26 With the finding that beta blockers are also in this category of therapeutic agents capable of reducing oxidative stress and ER stress, it is tempting to speculate that some of the known cardioprotection associated with these 2 distinct classes of agents with very diverse pharmacodynamics may be related to their effectiveness as ER stress attenuators. www.americantherapeutics.com
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Overall, this study indicates that ER stress, superoxide anion production, and cell death induced by 27.5 mM dextrose was inhibited by carvedilol, propranolol, and atenolol. However, it is noteworthy that although the antioxidative potential of beta blockers was confirmed in cell cultures, in healthy men, antihypertensive doses of carvedilol (25-mg twice a day) or 100-mg metoprolol twice a day exerted no specific inhibition of oxidative stress as measured by urinary excretion of 8-iso-PGF (2alpha) and 2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), and the plasma concentration of 3-nitrotyrosine and thiobarbituric acid-reactive substances.12 There are no in vivo studies on the effectiveness of beta blockers as ER stress reducers. Secondary prevention trials have shown that beta blockers are associated with 25% relative risk reduction in cardiovascular events.4 This favorable therapeutic effects of beta blockers are attributed mostly to the reduction in myocardial oxygen demand and heart rate. The beta blockers may also have a host of pleiotropic effects that may contribute to their salutary effects on survival. One such pleiotropic effect may well be their ability to reduce the oxidative stress and ER stress simultaneously. Therapeutic agents that can ameliorate both oxidative stress and ER stress in endothelial cells may well be proven to have clinically meaningful cardioprotective efficacy.
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Beta Blockers Suppress Dextrose-Induced Endoplasmic Reticulum Stress, Oxidative Stress, and Apoptosis in Human Coronary Artery Endothelial Cells Michael J. Haas, PhD, William Kurban, MD, Harshit Shah, MD, Luisa Onstead-Haas, MS, and Arshag D. Mooradian, MD*
Beta blockers are known to have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. To determine whether beta blockers can also prevent dextrose-induced endoplasmic reticulum (ER) stress in addition to their antioxidative effects, human coronary artery endothelial cells and hepatocyte-derived HepG2 cells were treated with 27.5 mM dextrose for 24 hours in the presence of carvedilol (a lipophilic beta blockers with alpha blocking activity), propranolol (a lipophilic nonselective beta blockers), and atenolol (a water-soluble selective beta blockers), and ER stress, oxidative, stress and cell death were measured. ER stress was measured using the placental alkaline phosphatase assay and Western blot analysis of glucose regulated protein 78, c-Jun-N-terminal kinase (JNK), phospho-JNK, eukaryotic initiating factor 2a (eIF2a), and phospho-eIF2a and measurement of X-box binding protein 1 (XBP1) mRNA splicing using reverse transcriptase-polymerase chain reaction. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A] pyrazin-3-one hydrochloride (MCLA) chemiluminescence. Cell viability was measured by propidium iodide staining method. The ER stress, SO production, and cell death induced by 27.5 mM dextrose were inhibited by all 3 beta blockers tested. The antioxidative and ER stress reducing effects of beta blockers were also observed in HepG2 cells. The salutary effects of beta blockers on endothelial cells in reducing both ER stress and oxidative stress may contribute to the cardioprotective effects of these agents. Keywords: beta blockers, endothelial cells, HepG2 cells, endoplasmic reticulum stress, cardiovascular disease
INTRODUCTION Cardiovascular disease is the leading cause of morbidity and mortality in people with diabetes mellitus.1,2 Only few classes of therapeutic agents are found to be effective in reducing cardiovascular event rates. These include antithrombotic agents, angiotensin converting enzyme Department of Medicine, University of Florida College of Medicine, Jacksonville, FL. Supported by Dean’s Fund Research Grant from the University of Florida College of Medicine, Jacksonville, FL. The authors have no conflicts of interest to declare. *Address for correspondence: Department of Medicine, University of Florida College of Medicine, 653-1 West Eighth St, Jacksonville, FL 32209. E-mail: [email protected]
inhibitors, or angiotensin receptor blockers, statins and beta blockers.3 Although the pharmacology of these agents is well characterized, the precise mechanisms underlying their cardioprotective properties are not entirely understood. The beneficial effects of beta blockers are attributed to their antiarrhythmogenic activity and their effectiveness in reducing myocardial oxygen demand through reducing the heart rate and myocardial contractility.4,5 However, known antiarrythmogenic drugs have either failed to improve patient outcomes and in some instances have increased the risk of mortality.6,7 Furthermore, calcium channel blockers that can reduce the heart rate and myocardial contractility have not been consistently associated with survival advantage, although they can be effective agents in controlling angina.8,9 Furthermore, ivabradine, an agent that selectively reduces the heart rate, was recently shown
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to have no benefit in patients with coronary artery disease.10 Thus, it is likely that the known cardioprotective properties of beta blockers are the results of biological changes that extend beyond their known effects on heart rate and myocardial contractility. Previously published studies have suggested that some beta blockers have antioxidative activity that can alter endothelial cell expression of vasoactive molecules and have effects on the proliferation and apoptosis of human coronary artery smooth muscle and endothelial cells.11–14 In the present communication, we report on an additional pleiotropic effect of beta blockers that may contribute to their cardioprotective effects, namely their ability to reduce ER stress.
MATERIALS AND METHODS Materials
pSEAP2-Control, containing a truncated placental alkaline phosphatase gene adjacent to the simian virus 40 early gene promoter, and 24 hours later treated with 5.5 or 27.5 mM dextrose with the carvedilol, propranolol, and atenolol indicated amounts or the solvent dimethyl sulfoxide (DMSO). After 24 hours, secreted alkaline phosphatase (SAP) activity was measured by mixing 25-mL of conditioned medium with 75-mL of dilution buffer, which was incubated at 65°C for 30 minutes in 1.5 mL microcentrifuge tubes. After cooling on ice for 3 minutes, 100 mL of CSPD was added to each tube. The samples were incubated at room temperature for 30 minutes, and SAP activity was measured with a Modulus luminometer (Promega, Madison, WI) and is expressed in relative light units (RLU). As a negative control, SAP activity was measured in cells transfected with pSEAP2-Basic, which contains the placental SAP gene but lacks a eukaryotic promoter to drive expression. The solvent DMSO (diluted 1: 1000) had no effect on SAP activity.
Carvedilol was obtained from Sigma–Aldrich (St. Louis, MO), atenolol was obtained from MP Biomedicals (Santa Ana, CA), and propranolol was obtained from Enzo Life Sciences (Farmingdale, NY). The chemiluminescent alkaline phosphatase substrate 3-(diethylamino)-1-(2,2-dimethyl-3-H-1-benzofuran7-yl)-propan-1-one (CSPD) was purchased from Clontech Laboratories Inc. (Mountain View, CA). Lipofectamine and the superoxide-reactive probe 2-Methyl-6-(4Methoxyphenyl)-3,7-Dihydroimidazo[1,2-A]pyrazin3-one hydrochloride (MCLA) were obtained from Life Technologies (Carlsbad, CA) All other chemicals were obtained from Sigma–Aldrich or Fisher Scientific (Pittsburgh, PA).
Superoxide (SO) generation was measured using MCLA chemiluminescence as previously described.17 HCAEC and HepG2 cells were treated with 5.5 or 27.5 mM dextrose with increasing amounts of carvedilol, atenolol, and propranolol in 6-well plates, and MCLA was added to a final concentration of 1 mmol/L in Hank balanced salt solution containing 1.26 mM CaCl2, 5.37 mM KCl, 0.44 mM KH2PO4, 0.49 mM MgCl2$6H2O, 0.41 mM MgSO4$7H2O, 136.7 mM NaCl, 4.2 mM NaHCO3, 0.34 mM Na2HPO4, and 5.5 mM D-glucose. Superoxide-induced chemiluminescence was measured with a luminometer and is expressed in RLU.
Cell culture
Western blotting
Human coronary artery endothelial cells (HCAEC) purchased from Life Technologies were maintained in phenol red-free Medium 200 (Life Technologies) containing 1% low serum growth supplement. The hepatoblastoma cell line HepG2 was purchased from American Type Culture Collection and were maintained in BioWhittaker Dulbecco modified essential medium (Fisher Scientific) containing 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 units per milliliter of penicillin. The HCAEC and HepG2 cells were maintained in a humidified incubator at 37°C and 5% CO2.
HCAEC were treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, and propranolol and after 24 hours later, protein extracts were prepared by suspending the cells in 200 mL of lysis buffer (12.5% glycerol, 40 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 8.0), 5% 2-mercaptoethanol, and 1% sodium dodecylsulfate). Protein content was measured using the Bradford assay,18 and 50 mg of protein extract was fractionated by electrophoresis on a 10%-sodium dodecylsulfate polyacrylamide gel.19 After transfer to nitrocellulose,20 the membrane was blocked with 10% newborn calf serum in Tris-buffered saline/Tween 20 (TBST; 50 mM Tris-Cl, 150 mM NaCl, and 0.5% Tween 20, pH 7.4) and incubated overnight with antibodies to glucose regulated protein 78 (GRP78), c-Jun-N-terminal kinase (JNK), phospho-JNK (all from Cell Signaling Technology, Danvers, MA) (all diluted 1:750), or eukaryotic
ER stress measurement ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay15,16 (Great EscAPe; Clontech Laboratories Inc, Mountain View, CA). Briefly, HCAEC and HepG2 cells at 70%–80% confluence in 6-well plates were transfected with the plasmid American Journal of Therapeutics (2015) 0(0)
Superoxide generation
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Beta Blockers Suppress ER Stress
initiating factor 2a (eIF2a), and phospho-eIF2a (diluted 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Southern Biotech., Birmingham, AL) (diluted 1:5000), and binding was detected by enhanced chemiluminescence with reagents from Pierce Biotechnology (Rockford, IL). X-box binding protein 1 (XBP1) mRNA splicing Total RNA was isolated from endothelial cells treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, and propranolol for 24 hours. One microgram of RNA was reverse transcribed, and 100 ng of complementary DNA was used in each polymerase chain reaction with the following primers. XBP1 forward, 59-TTA CGA GAG AAA ACT CAT GGC C-39; XBP1 reverse, 59-GGG TCC AAG TTG TCC AGA ATG C-39. After polymerase chain reaction, the samples were fractionated by electrophoresis on a 3% agarose gel in Tris-borate-EDTA [TBE; 89 mM Tris-Cl, 89 mM boric acid, 2 mM ethylenediamine tetraacetic acid (EDTA)]. The gel was stained with ethidium bromide and photographed under ultraviolet light. Measurement of endothelial cell viability HCAEC were cultured in 6-well plates and treated with 5.5 or 27.5 mM dextrose with and without 10 mM carvedilol, propranolol, or atenolol. After 24 and 48 hours, propidium iodide (PI) was added at a final concentration of 10 mg/mL, and the cultures were incubated with the cells for 5 minutes as previously described.21 The cells were visualized under ultraviolet light (340–380 nM), and the number of PI-positive cells was counted in 4 fields, over 150 cells per field. Statistical analysis All results are expressed as mean 6 SD. Analysis of variance followed by the Newman–Keuls procedure for subgroup analysis was performed using the statistical package Statistica for Windows (Statsoft Inc, Tulsa, OK). Significance was defined as a 2-tailed P , 0.05. The half maximal effective concentration (EC50) for carvedilol, atenolol, and propranolol effects on SO generation, and ER stress in HCAEC and HepG2 cells was obtained from nonlinear regression of the dose response curves using GraphPad Prism 5 (La Jolla, CA).
RESULTS Effect of beta blockers on dextrose-induced ER stress in HCAEC The SAP activity decreased from 248,263 6 20,483 RLU in control cells treated with 5.5 mM dextrose www.americantherapeutics.com
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to 164,775 6 13,629 RLU in cells treated with 27.5 mM dextrose (P , 0.001), indicating increased ER stress (Figure 1A). Addition of carvedilol increased SAP activity to 160,517 6 19,544, 201,609 6 18,164, 261,546 6 16,189 RLU in cells treated with 0.1, 1.0, and 10 mM carvedilol (nonsignificant [NS], P , 0.05, and P , 0.001, respectively) (Figure 1A). Treatment of cells with 0.1, 1.0, and 10 mM atenolol increased SAP activity from a baseline of 177,627 6 110,772 RLU to 192,670 6 13,154, 213,978 6 9826, and 266,393 6 11,187 RLU (NS, P , 0.01, and P , 0.001, respectively) (Figure 1B), and treatment of cells with 0.1, 1.0, and 10 mM propranolol increased SAP activity from a baseline of 134,424 6 12,762 RLU to 133,003 6 7353, 159,604 6 16,703, and 177,214 6 1152 RLU (NS, P , 0.05, and P , 0.01, respectively) (Figure 1C). Addition of carvedilol, atenolol, or propranolol (Figures 2A–C) had no effect on SAP activity in HCAEC treated with 5.5 mM dextrose (all NS). These results indicate that all 3 beta blockers inhibit dextrose-induced ER stress in HCAEC with an EC50 of 1.5, 1.6, and 1.1 mM for carvedilol, atenolol, and propranolol, respectively (Table 1). Effect of beta blockers on ER stress markers Treatment of HCAEC with 27.5 mM dextrose increased GRP 78 expression, eIF2a phosphorylation, and JNK phosphorylation relative to cells treated with 5.5 mM dextrose (Figure 2A). In contrast, when HCAEC were treated with 10 mM carvedilol, 10 mM atenolol, and 10 mM propranolol, and 27.5 mM dextrose, GRP 78 expression was similar to HCAEC treated with 5.5 mM dextrose (Figure 2A). Likewise, eIF2a and JNK phosphorylation in carvedilol-, atenolol-, and propranolol-treated cells exposed to 27.5 mM dextrose was similar to eIF2a and JNK phosphorylation in HCAEC exposed to 5.5 mM dextrose (Figure 2A). Splicing of the XBP1 mRNA is also associated with induction of ER stress.16 Treatment of HCAEC with 27.5 mM dextrose induced XBP1 mRNA splicing (Figure 2B, lanes 2 and 3). Addition of carvedilol (10 mM), atenolol (10 mM), or propranolol (10 mM) inhibited XBP1 mRNA splicing (Figure 2B, lanes 4–6). These results further confirm that all 3 beta blockers examined inhibit ER stress in HCAEC. Effect of beta blockers on SO generation in HCAEC Treating HCAEC with high ambient dextrose concentration increased SO generation from a baseline of 5102 6 197 RLU to 6376 6 146 RLU in cells exposed to 27.5 mM dextrose (P , 0.009) (Figure 3A). Addition of 0.1, 1.0, and 10 mM carvedilol decreased SO American Journal of Therapeutics (2015) 0(0)
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FIGURE 1. The effect of 0, 0.1, 1.0, and 10 mM carvedilol, atenolol, and propranolol on dextrose-induced ER stress in HCAEC treated with 5.5 or 27.5 mM dextrose. ER stress (inverse of SAP activity) was measured 24 hours after treating the cells. (A) Carvedilol (CV). N 5 6; *P , 0.001, relative to cells treated with 5.5 mM dextrose; †P , 0.05 and P , 0.001, relative to cells treated with 27.5 mM dextrose. (B) Atenolol (Aten.). N 5 6; *P , 0.003, relative to cells treated with 5.5 mM dextrose; †P , 0.01 and P , 0.001, relative to cells treated with 27.5 mM dextrose. (C) Propranolol (Prop.). N 5 6; *P , 0.006, relative to cells treated with 5.5 mM dextrose; †P , 0.05 and P , 0.01, relative to cells treated with 27.5 mM dextrose. †P-values shown are for 0.1, 1.0, and 10 mM concentration of the agents used, respectively.
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FIGURE 2. The effect of carvedilol, atenolol, and propranolol on ER stress markers in human coronary artery endothelial cells. (A) Cells were treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, atenolol, or propranolol for 24 hours. GRP 78, phospho-eIF2a, eIF2a, phospho-JNK, and JNK were measured by Western blotting. (B) XBP1 mRNA splicing was examined by reverse-transcription polymerase chain reaction. Treatment with 27.5 mM dextrose increased GRP 78 levels and phospho-eIF2a and phospho-JNK levels (A) and splicing of the XBP1 mRNA (B). Treatment with carvedilol, atenolol, and propranolol decreased GRP 78, phospho-eIF2a, phospho-JNK levels (A) and decreased the amount of the XBP1 spliced mRNA levels (B).
generation to 5558 6 186, 5055 6 180, and 5212 6 128 RLU, respectively (P , 0.004, 0.006, and 0.0005, respectively) (Figure 3A). Treating cells with 0.1, 1.0, and 10 mM atenolol decreased SO generation in HCAEC exposed to 27.5 mM dextrose from a baseline of 6335 6 633 RLU to 6244 6 765, 5076 6 405, and 3599 6 492 RLU, respectively (NS, P , 0.04 and 0.004, respectively) (Figure 3B), and treating cells with 0.1, 1.0, and 10 mM propranolol reduced SO generation www.americantherapeutics.com
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Table 1. Effect of carvedilol, atenolol, and propranolol on SO generation and ER stress in HCAEC and hepatomaderived (HepG2) cells. Carvedilol Atenolol Propranolol EC50, mM EC50, mM EC50, mM HCAEC SO generation ER stress HepG2 SO generation ER stress
0.6 1.5
1.3 1.6
0.8 1.1
0.1 2.3
1.4 1.8
2.2 2.0
EC50 values were calculated from the data in Figures 1, 2 and dose response curves performed with HepG2 cells.
in HCAEC exposed to 27.5 mM dextrose from a baseline of 11,805 6 1232 RLU to 11,799 6 964, 9104 6 1436, and 6612 6 1325 RLU, respectively (NS, NS, and P , 0.004, respectively) (Figure 3C). Addition of carvedilol, atenolol, or propranolol (Figures 3A–C) had no effect on ROS generation in HCAEC treated with 5.5 mM dextrose (all NS). These results indicate that all 3 beta blockers inhibit dextrose-induced SO generation in HCAEC with an EC50 of 0.6, 1.3, and 0.8 mM for carvedilol, atenolol, and propranolol, respectively (Table 1). Effect of beta blockers on endothelial cell viability Long-term exposure of HCAEC to hyperglycemic conditions leads to enhanced cell death.16,17 To determine whether carvedilol, atenolol, and propranolol can prevent dextrose-induced endothelial cell death, HCAEC were treated with 5.5 and 27.5 mM dextrose for 24 (Figure 4A) and 48 hours (Figure 4B) in the presence or absence of 10 mM carvedilol, 10 mM atenolol, or 10 mM propranolol. Addition of carvedilol, atenolol, and propranolol decreased the number of PI staining cells at both 24 and 48 hours. Effect of beta blockers on dextrose-induced ER stress and SO generation in HepG2 cells To determine whether carvedilol, atenolol, and propranolol inhibit ER stress and SO generation in nonendothelial cells, HepG2 cells were treated with 5.5 and 27.5 mM dextrose, and ER stress (Figure 5A) and SO generation (Figure 5B) were measured as described above. High concentrations of dextrose reduced SAP expression from 108,893 6 6956 RLU to 82,502 6 2837 RLU (P , 0.004). Addition of 10 mM carvedilol, 10 mM atenolol, and 10 mM propranolol increased SAP expression to 10,697 6 4796, www.americantherapeutics.com
FIGURE 3. The effect of 0, 0.1, 1.0, and 10 mM carvedilol, atenolol, and propranolol on dextrose-induced superoxide (SO) generation in HCAEC treated with 5.5 or 27.5 mM dextrose. The SO generation was measured 24 hours after treatment of cells by MCLA chemiluminescence. (A) Carvedilol (CV). N 5 6; *P , 0.009, relative to cells treated with 5.5 mM dextrose; †P , 0.004, P , 0.006, and P , 0.0005, relative to cells treated with 27.5 mM dextrose. (B) Atenolol (Aten.). N 5 6; *P , 0.004, relative to cells treated with 5.5 mM dextrose; †P , 0.04 and P , 0.004, relative to cells treated with 27.5 mM dextrose. (C) Propranolol (Prop.). N 5 6; *P , 0.002, relative to cells treated with 5.5 mM dextrose; †P , 0.004, relative to cells treated with 27.5 mM dextrose. †P values shown are for 0.1, 1.0, and 10 mM concentration of the agents used, respectively.
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FIGURE 4. The effect of carvedilol, atenolol, and propranolol on dextrose-induced human coronary artery endothelial cell viability. Cells were treated with either 5.5 or 27.5 mM dextrose for 24 hours (A) or 48 hours (B) in the presence of 10 mM carvedilol (CV), 10 mM atenolol (Aten.), or 10 mM propranolol (Prop.). Cell viability was measured by staining with PI, and dying cells were counted under fluorescent illumination. Treatment of cells with 27.5 mM dextrose increased the number of PI-positive cells at both 24 and 48 hours. Addition of carvedilol, atenolol, and propranolol decreased the number of PI-positive cells at both 24 and 48 hours. (A) *P , 0.07, relative to cells treated with 5.5 mM dextrose; †P , 0.05, P , 0.06, and P , 0.07, relative to cells treated with 27.5 mM dextrose. (B) *P , 0.01, relative to cells treated with 5.5 mM dextrose; †P , 0.008, P , 0.005, and P , 0.09, relative to cells treated with 27.5 mM dextrose. †P values shown are for carvedilol, atenolol, and propranolol, respectively.
FIGURE 5. The effect of carvedilol, atenolol, and propranolol on superoxide (SO) generation and ER stress in HepG2 cells. (A) HepG2 cells were treated with 5.5 and 27.5 mM dextrose with or without 10 mM carvedilol (CV), 10 mM atenolol (Aten.), or 10 mM propranolol (Prop.), and SO generation was measured. (B) HepG2 cells were transfected with pSEAP-Control and 24 hours later treated with 5.5 or 27.5 mM dextrose with or without 10 mM carvedilol, 10 mM atenolol, or 10 mM propranolol. After 24 hours, SAP activity was measured. Carvedilol, atenolol, and propranolol decreased SO production and ER stress in HepG2 cells treated with 27.5 mM dextrose. (A) N 5 6; *P , 0.0004, relative to cells treated with 5.5 mM dextrose; †P , 0.002, P , 0.008, P , 0.005, relative to cells treated with 27.5 mM dextrose. (B) N 5 6; *P , 0.002, relative to cells treated with 5.5 mM dextrose; †P , 0.0005, P , 0.001, P , 0.002, relative to cells treated with 27.5 mM dextrose. †P values shown are for carvedilol, atenolol, and propranolol, respectively.
110,938 6 4674, and 107,649 6 7210 RLU, respectively (P , 0.002, 0.0008, and 0.005, respectively). High ambient concentrations of dextrose induced SO generation in HepG2 cells (Figure 5B) from 3664 6 254 RLU to 4926 6 175 RLU (P , 0.002). Addition of 10 mM carvedilol, 10 mM atenolol, and 10 mM
propranolol decreased SO generation to 3638 6 117, 3788 6 160, and 3747 6 214 RLU, respectively (P , 0.0005, 0.001, and 0.002, respectively). In these cells, the EC50 for carvedilol, atenolol, and propranolol in reducing SO generation was 0.1, 1.4, and 2.2 mM, respectively, and for ER stress reduction, the EC50s were
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Beta Blockers Suppress ER Stress
2.3, 1.8, and 2.0 mM, respectively (Table 1). These results indicate that the 3 beta blockers tested inhibit ER stress and oxidative stress in HepG2 cells and in HCAEC.
DISCUSSION Human endothelial cells in culture contain ample betaadrenergic receptors, mostly the beta 2-subtype.22 These receptors are implicated in the regulation of renin–angiotensin system of the endothelial cells23 and in the production of other vasoactive peptides such as endothelin 1.11,14 In addition, previous studies have shown that beta-adrenergic receptor blockers, notably carvedilol, have a potent antioxidant activity11,12 and may also have favorable effects on cell survival.11,13 This study confirms the previously reported antioxidative properties of beta blockers and more importantly demonstrates for the first time that beta blockers as a class may be effective in reducing the ER and oxidative stress simultaneously. The 3 agents tested were effective in both endothelial cells and the HepG2 cells, although the differences in the potency of these 3 agents were less apparent in the HepG2 cells. Analysis of concentration–response curves reveals that in endothelial cells, carvedilol is the most potent of the 3 as to its antioxidative potential with an EC50 that as 25%–50% lower than that of propranolol and atenolol, respectively, whereas in HepG2 cells, the EC50 of carvedilol for inhibition of SO generation was 10- to 20-fold lower than of atenolol and propranolol (Table 1). In contrast, the potency of these 3 beta blockers in reducing ER stress was comparable, and overall, they appeared to be more potent ER stress suppressors in endothelial cells than in HepG2 cells (Table 1). Although there may be a link between oxidative stress and ER stress, the 2 pathways can be dissociated and independent.24,25 The proximal signals for these 2 stresses are generated by different intermediaries of glucose metabolism,16 and whereas antioxidant vitamins are effective in reducing oxidative stress that do not attenuate ER stress.17 Thus, there seems to be limited classes of agents that are effective in reducing oxidative stress and ER stress simultaneously. In this category of dual stress reducers, statins have been shown to be effective in endothelial cells and in HepG2 cells.26 With the finding that beta blockers are also in this category of therapeutic agents capable of reducing oxidative stress and ER stress, it is tempting to speculate that some of the known cardioprotection associated with these 2 distinct classes of agents with very diverse pharmacodynamics may be related to their effectiveness as ER stress attenuators. www.americantherapeutics.com
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Overall, this study indicates that ER stress, superoxide anion production, and cell death induced by 27.5 mM dextrose was inhibited by carvedilol, propranolol, and atenolol. However, it is noteworthy that although the antioxidative potential of beta blockers was confirmed in cell cultures, in healthy men, antihypertensive doses of carvedilol (25-mg twice a day) or 100-mg metoprolol twice a day exerted no specific inhibition of oxidative stress as measured by urinary excretion of 8-iso-PGF (2alpha) and 2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), and the plasma concentration of 3-nitrotyrosine and thiobarbituric acid-reactive substances.12 There are no in vivo studies on the effectiveness of beta blockers as ER stress reducers. Secondary prevention trials have shown that beta blockers are associated with 25% relative risk reduction in cardiovascular events.4 This favorable therapeutic effects of beta blockers are attributed mostly to the reduction in myocardial oxygen demand and heart rate. The beta blockers may also have a host of pleiotropic effects that may contribute to their salutary effects on survival. One such pleiotropic effect may well be their ability to reduce the oxidative stress and ER stress simultaneously. Therapeutic agents that can ameliorate both oxidative stress and ER stress in endothelial cells may well be proven to have clinically meaningful cardioprotective efficacy.
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