Inhibition of Pro-Inflammatory Cytokine Secretion by Select Antioxidants in Human Coronary Artery Endothelial Cells.

Inflammatory and oxidative stress in endothelial cells are implicated in the pathogenesis of premature atherosclerosis in diabetes. To determine whether high-dextrose concentrations induce the expression of pro-inflammatory cytokines, human coronary artery endothelial cells (HCAEC) were exposed to either 5.5 or 27.5 mM dextrose for 24-hours and interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α (TNF α) levels were measured by enzyme immunoassays. To determine the effect of antioxidants on inflammatory cytokine secretion, cells were also treated with α-tocopherol, ascorbic acid, and the glutathione peroxidase mimetic ebselen. Only the concentration of IL-1ß in culture media from cells exposed to 27.5 mM dextrose increased relative to cells maintained in 5.5 mM dextrose. Treatment with α-tocopherol (10, 100, and 1,000 µM) and ascorbic acid (15, 150, and 1,500 µM) at the same time that the dextrose was added reduced IL-1ß, IL-6, and IL-8 levels in culture media from cells maintained at 5.5 mM dextrose but had no effect on IL-1ß, IL-6, and IL-8 levels in cells exposed to 27.5 mM dextrose. However, ebselen treatment reduced IL-1ß, IL-6, and IL-8 levels in cells maintained in either 5.5 or 27.5 mM dextrose. IL-2 and TNF α concentrations in culture media were below the limit of detection under all experimental conditions studied suggesting that these cells may not synthesize detectable quantities of these cytokines. These results suggest that dextrose at certain concentrations may increase IL-1ß levels and that antioxidants have differential effects on suppressing the secretion of pro-inflammatory cytokines in HCAEC.

The Effects of Known Cardioprotective Drugs on Proinflammatory Cytokine Secretion From Human Coronary Artery Endothelial Cells.

BACKGROUND: Endothelial cell dysfunction in diabetes is involved in the pathogenesis and progression of premature atherosclerosis. High-dextrose has been shown to induce both oxidative stress and endoplasmic reticulum stress in cultured human coronary artery endothelial cells (HCAEC). STUDY QUESTION: To determine whether or not several classes of cardioprotective drugs inhibit proinflammatory cytokine expression by HCAEC. MEASURES AND OUTCOMES: To determine the effects of high dextrose on expression of proinflammatory cytokines by HCAEC, cells were treated with either 5.5 mM or 27.5 mM dextrose for 24 hours and interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α were measured by enzyme immunoassay in the presence or absence of known cardioprotective drugs, including select ß-blockers, statins, and renin-angiotensin system inhibitors. RESULTS: IL-1ß levels increased significantly in cells treated with high dextrose; however, IL-6 and IL-8 levels did not change. Treatment of cells with carvedilol, atenolol, and propranolol decreased levels of all 3 cytokines in cells exposed to either 5.5 or 27.5 mM dextrose. Similar effects on IL-1ß, IL-6, and IL-8 levels were observed when cells were treated with simvastatin, pravastatin, and the renin-angiotensin system inhibitors spironolactone, captopril, lisinopril, candesartan, and losartan. No Il-2 or tumor necrosis factor α expression was observed in any of the experiments indicating that HCAEC do not express these cytokines. CONCLUSIONS: We conclude that each of the classes of drugs tested possess pleiotropic anti-inflammatory activities and are effective in both low- and high-dextrose-treated cells.

Transcription factor EB protects against endoplasmic reticulum stress in human coronary artery endothelial cells.

Oxidative stress and endoplasmic reticulum (ER) stress promote atherogenesis while transcription factor EB (TFEB) inhibits atherosclerosis. Since reducing oxidative stress with antioxidants have failed to reduce atherosclerosis possibly because of aggravation of ER stress, we studied the effect of TFEB on ER stress in human coronary artery endothelial cells. ER stress was measured using the secreted alkaline phosphatase assay. Expression and phosphorylation of key mediators of unfolded protein response (UPR). TFEB, inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phospho-PERK, and activating transcription factor 6 (ATF6) expression were measured by Western blot. The effect of TFEB gain- and loss-of-function on ER stress were assessed with a plasmid expressing a constitutively active form of TFEB and via siRNA-mediated silencing, respectively. Treatment with tunicamycin (TM) and thapsigargin (TG) increased TFEB expression by 42.8% and 42.3%, respectively. In HCAEC transfected with the TFEB siRNA, treatment with either TM, TG or high-dextrose increased IRE1α and PERK phosphorylation and ATF6 levels significantly more compared to cells transfected with the control siRNA and treated similarly. Furthermore, transient transfection with a plasmid expressing a constitutively active form of TFEB reduced ER stress. Increased expression of TFEB inhibited ER stress in HCAEC treated with pharmacologic (TM and TG) and physiologic (high-dextrose) ER stress inducers, while TFEB knockout aggravated ER stress caused by these ER stress inducers. TFEB-mediated ER stress reduction may contribute to its anti-atherogenic effects in HCAEC and may be a novel target for drug development.

The effect of nicotine and dextrose on endoplasmic reticulum stress in human coronary artery endothelial cells.

Cigarette smoking is one of the major causes of coronary artery disease (CAD) as is diabetes. However, nicotine has been generally regarded as safe and is used in smoking cessation programs. This presumption of nicotine safety was examined in human coronary artery endothelial cells (HCAEC). Endoplasmic reticulum (ER) stress was measured using the secreted alkaline phosphatase (SAP) assay. The ER stress markers inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK, activating transcription factor 6 (ATF6), and glucose-related protein 78 (GRP78) were measured by western blot. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Intact and cleaved caspase 3, BH3 interacting-domain death agonist (BID), and B-cell lymphoma 2 (Bcl2) were measured by western blot. In cells transfected with the SAP expression plasmid, treatment with nicotine resulted in a dose-dependent decrease in SAP expression with no noticeable toxicity. Nicotine (10 nM) also increased IRE1α and PERK phosphorylation, and ATF6 and GRP78 expression. Although nicotine at concentrations up to 10 µM did not cause cell death, treatment of HCAEC with 10 nM nicotine in the presence of 13.8 mM dextrose aggravated ER stress, increased cell death, increased cleaved caspase 3 and BID, and decreased BCL2. Nicotine at concentrations commonly achieved in nicotine-replacement therapy (NRT) significantly increased ER stress in HCAEC and aggravated dextrose-induced ER stress and cell apoptosis. People using electronic cigarettes and on NRT may be at increased risk for CAD.

High-throughput analysis identifying drugs that reduce oxidative and ER stress in human coronary artery endothelial cells.

Endoplasmic reticulum (ER) stress as well as oxidative stress have been shown to play important roles in metabolic and cardiovascular disease, and drugs that counteract the effects of ER and oxidative stresses may be clinically useful. To identify novel compounds that ameliorate ER and oxidative stresses, we screened two drug libraries purchased from Evotec, San Francisco, CA; the NIH clinical collection 1 (446 compounds) and the NIH clinical collection 2 (281 compounds). Human coronary artery endothelial cells (HCAEC) were tested for ER and oxidative stress. ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (SAP) assay. The cells were transfected with the plasmid pSAP2.Control, expressing a heat-resistant form of SAP, and treated with the ER stress inducer tunicamycin in the presence or absence of each of the various compounds for 24-h, at which time SAP activity was measured. Compounds exhibiting significant increases in SAP activity (41 compounds out of a total of 727 tested; 5.6%) were then assayed for their ability to suppress superoxide (SO) anion generation in cells treated with 27.5 mM dextrose. SO generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence. Of the 41 compounds identified as ER stress reducers, only 33 (80.5%) suppressed dextrose-induced SO anion generation. Interestingly, 51% of the compounds found to be dual-stress modifiers consisted of cardioprotective drugs, including statins, angiotensin receptor blockers, angiotensin-converting enzyme inhibitors as well as ß-blockers. Future studies to validate the clinical effectiveness of these agents remain to be performed in pre-clinical and clinical trials.