Cardioprotective antihyperglycemic drugs ameliorate endoplasmic reticulum stress.

Cellular stress, notably oxidative, inflammatory, and endoplasmic reticulum (ER) stress, is implicated in the pathogenesis of cardiovascular disease. Modifiable risk factors for cardiovascular disease such as diabetes, hypercholesterolemia, saturated fat consumption, hypertension, and cigarette smoking cause ER stress whereas currently known cardioprotective drugs with diverse pharmacodynamics share a common pleiotropic effect of reducing ER stress. Selective targeting of oxidative stress with known antioxidative vitamins has been ineffective in reducing cardiovascular risk. This "antioxidant paradox" is partially attributed to the unexpected aggravation of ER stress by the antioxidative agents used. In contrast, some of the contemporary antihyperglycemic drugs inhibit both oxidative stress and ER stress in human coronary artery endothelial cells. Unlike sulfonylureas, meglitinides, α glucosidase inhibitors, and thiazolidinediones, metformin, glucagon-like peptide 1 receptor agonists, and sodium-glucose cotransporter 2 inhibitors are the only antihyperglycemic drugs that reduce ER stress caused by pharmacological agents (tunicamycin) or hyperglycemic conditions. Clinical trials with selective ER stress modifiers are needed to test the suitability of ER stress as a therapeutic target for cardiovascular disease.

Naturally occurring rare sugars are free radical scavengers and can ameliorate endoplasmic reticulum stress.

Because of potential use of naturally occurring rare sugars as sweeteners, their effect on superoxide (SO), hydroxyl and peroxyl radicals and endoplasmic reticulum (ER) stress was examined in human coronary artery endothelial cells. 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. Phycoerythrin fluorescence based assay was used to monitor scavenging activity of sugars in the presence of hydroxyl or peroxyl radical generators [CuSO4 and azobis (2 amidinopropane) hydrochloride respectively]. Measurements were made in relative light units (RLU). ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (SAP) assay and by Western blot analysis of the expression and phosphorylation of key proteins in the unfolded protein response, namely CHOP47, eIF2α and JNK1. D-Glucose (27.5 mM) increased SO generation (5536 ± 283 vs. 2963 ± 205 RLU in controls; p < 0.0007) and decreased SAP secretion (73411 ± 3971 vs. 101749 ± 7652 RLU in controls; p < 0.005) indicating ER stress. Treatment of cells with 5.5 or 27.5 mM of D-allulose, D-allose, D-sorbose and D-tagatose reduced SO generation (all p < 0.05). This could not be attributed to inhibition of cellular uptake of dextrose by the rare sugars tested. In a cell free system, all four rare sugars had significantly more SO, hydroxyl and peroxyl radical scavenging activity compared to dextrose (all p < 0.01). Treatment of cells with rare sugars reduced ER stress. However, unlike other three rare sugars, D-sorbose did not inhibit tunicamycin-induced eIF2α phosphorylation. Naturally occurring rare sugars are free radical scavengers and can reduce ER stress.

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.

Inhibition of ABCA1 Protein Expression and Cholesterol Efflux by TNF α in MLO-Y4 Osteocytes.

Hip fracture and myocardial infarction cause significant morbidity and mortality. In vivo studies raising serum cholesterol levels as well as pro-inflammatory cytokines such as TNF α manifest bone loss and atherosclerotic vascular disease, suggesting that abnormalities of cholesterol transport may contribute to osteoporosis. We used the mouse osteocyte cell line (MLO-Y4) to investigate the effects of TNF α on the expression of cholesterol acceptor proteins such as apolipoprotein A-I (apo A-I) and apolipoprotein E (apo E), as well as on the cholesterol transporters ATP-binding cassette-1 (ABCA1), scavenger receptor class B type 1 (SRB1), and cluster of differentiation 36 (CD36). MLO-Y4 cells do not express apo A-I or apo E; however, they do express all three cholesterol transporters (ABCA1, SRB1, and CD36). Treatment of MLO-Y4 cells with TNF α had no effect on SRB1, CD36, and osteocalcin levels; however, TNF α reduced ABCA1 protein levels in a dose-dependent manner and cholesterol efflux to apo A-I. Interestingly, TNF α treatment increased ABCA1 promoter activity and ABCA1 mRNA levels, and increased liver X receptor α protein expression, but had no effect on retinoid X receptor α and retinoic acid receptor α levels. Pharmacological inhibition of p38 mitogen-activated protein (MAP) kinase, but not c-jun-N-terminal kinase 1 or mitogen-activated protein kinase (MEK), restored ABCA1 protein levels in TNF α-treated cells. These results suggest that pro-inflammatory cytokines regulate cholesterol metabolism in osteocytes in part by suppressing ABCA1 levels post-translationally in a p38 MAP kinase-dependent manner.

Beta Blockers Suppress Dextrose-Induced Endoplasmic Reticulum Stress, Oxidative Stress, and Apoptosis in Human Coronary Artery Endothelial Cells.

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 2α (eIF2α), and phospho-eIF2α 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.

Statins Prevent Dextrose-Induced Endoplasmic Reticulum Stress and Oxidative Stress in Endothelial and HepG2 Cells.

Statins have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. However, antioxidant vitamins, unlike statins, are not as cardioprotective, and this paradox has been explained by failure of vitamin antioxidants to ameliorate endoplasmic reticulum (ER) stress. To determine whether statins prevent dextrose-induced ER stress in addition to their antioxidative effects, human umbilical vein endothelial cells and HepG2 hepatocytes were treated with 27.5 mM dextrose in the presence of simvastatin (lipophilic statin that is a prodrug) and pravastatin (water-soluble active drug), and oxidative stress, ER stress, and cell death were measured. Superoxide generation was measured using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride. ER stress was measured using the placental alkaline phosphatase assay and Western blot of glucose-regulated protein 75, c-jun-N-terminal kinase, phospho-JNK, eukaryotic initiating factor 2α and phospho-eIF2α, and X-box binding protein 1 mRNA splicing. Cell viability was measured by propidium iodide staining. Superoxide anion production, ER stress, and cell death induced by 27.5 mM dextrose were inhibited by therapeutic concentrations of simvastatin and pravastatin. The salutary effects of statins on endothelial cells in reducing both ER stress and oxidative stress observed with pravastatin and the prodrug simvastatin suggest that the effects may be independent of cholesterol-lowering activity.

Inhibition of apolipoprotein A-I expression by TNF-alpha in HepG2 cells: requirement for c-jun.

Tumor necrosis factor alpha (TNF α) signals in part through the mitogen activated protein (MAP) kinase c-jun-N-terminal kinase (JNK). Activation of JNK has been shown to promote insulin resistance and dyslipidemia, including reductions in plasma high-density lipoprotein (HDL) and apolipoprotein A-I (apo A-I). To examine how TNF α-mediated JNK activation inhibits hepatic apo A-I production, the effects of c-jun activation on apo A-I gene expression were examined in HepG2 cells. Apo A-I gene expression and promoter activity were measured by Northern and Western blotting and transient transfection. Transient transfection and siRNA were used to specifically over-express or knockout c-jun, c-jun-N-terminal kinase-1 and -2 (JNK1 and JNK2, respectively) and mitogen-activated protein kinase-4 (MKK4). TNF α-treatment of HepG2 cells induced rapid phosphorylation of c-jun on serine 63. In cells treated with phorbol-12-myristate-13-acetate (PMA), apo A-I gene promoter activity was inhibited and apo A-I mRNA content and apo A-I protein secretion decreased. Likewise, over-expression of JNK1 and JNK2 inhibited apo A-I promoter activity. Over-expression of constitutively active MKK4, an upstream protein kinase that directly activates JNK, also inhibited apo A-I promoter activity, while over-expression of a dominant-negative MKK4 de-repressed apo A-I promoter activity in TNF α-treated cells. Inhibition of c-jun synthesis using siRNA but not a control siRNA prevented TNF α-mediated inhibition of apo A-I. These results suggest that the MKK4/JNK/c-jun signaling pathway mediates TNF α-dependent inhibition of apo A-I synthesis.

Identification of ATP8B1 as a blood-brain barrier-enriched protein.

In order to define the molecular anatomy of the blood-brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.

The effect of black seed (Nigella sativa) extract on FOXO3 expression in HepG2 cells.

Black seed extracts are known to alter cellular metabolism through multiple signaling pathways. Since Forkhead box transcription factor 3 (FOXO3) has a significant role in regulating cellular metabolism, the effect of lipid extracts of black seed (Sativa nigella) on FOXO3 levels and AKT and 5-AMP activated protein kinase α (AMPKα) signaling was measured in HepG2 hepatoma cells. FOXO3 levels, phosphorylation, and nuclear exclusion were measured by Western blot, as were AKT and AMPK expression and activity using phosphorylation-specific antibodies. Apolipoprotein A-I expression, a black seed-responsive gene, was measured by Western blot. Treatment with black seed extract increased FOXO3 phosphorylation and decreased its expression. In contrast to control cells where FOXO3 was located primarily in the nucleus, in black seed-treated HepG2 cells, FOXO3 was localized primarily to the cytoplasm. These changes in FOXO3 phosphorylation, expression, and localization were accompanied by increased AKT activity. Black seed also decreased AMPKα activity but increased AMPKα expression. Lipid extracts from black seeds inhibit FOXO3 activity and thereby modulate the expression of FOXO3-dependent genes.

Induction of apolipoprotein A-I gene expression by black seed (Nigella sativa) extracts.

CONTEXT: Black seed [Nigella sativa L. (Ranunculaceae)] has been shown in animal models to lower serum cholesterol levels. OBJECTIVES: In order to determine if extracts from black seed have any effects on high-density lipoprotein (HDL), we characterized the effects of black seed extract on apolipoprotein A-I (apo A-I) gene expression, the primary protein component of HDL. MATERIALS AND METHODS: Hepatocytes (HepG2) and intestinal cells (Caco-2) were treated with black seed extracts, and Apo A-I, peroxisome proliferator-activated receptor α (PPARα), and retinoid-x-receptor α (RXRα) were measured by Western blot analysis. Apo A-I mRNA levels were measured by quantitative real-time polymerase chain reaction and apo A-I gene transcription was measured by transient transfection of apo A-I reporter plasmids. RESULTS: Extracts from black seeds significantly increased hepatic and intestinal apo A-I secretion, as well as apo A-I mRNA and gene promoter activity. This effect required a PPARα binding site in the apo A-I gene promoter. Treatment of the extract with either heat or trypsin had no effect on its ability to induce apo A-I secretion. Treatment with black seed extract induced PPARα expression 9-fold and RXRα expression 2.5-fold. Furthermore, the addition of PPARα siRNA but not a control siRNA prevented some but not all the positive effects of black seed on apo A-I secretion. DISCUSSION: Black seed extract is a potent inducer of apo A-I gene expression, presumably by enhancing PPARα/RXRα expression. CONCLUSIONS: We conclude that black seed may have beneficial effects in treating dyslipidemia and coronary heart disease.

Potential Therapeutic Agents That Target ATP Binding Cassette A1 (ABCA1) Gene Expression.

The cholesterol efflux protein ATP binding cassette protein A1 (ABCA) and apolipoprotein A1 (apo A1) are key constituents in the process of reverse-cholesterol transport (RCT), whereby excess cholesterol in the periphery is transported to the liver where it can be converted primarily to bile acids for either use in digestion or excreted. Due to their essential roles in RCT, numerous studies have been conducted in cells, mice, and humans to more thoroughly understand the pathways that regulate their expression and activity with the goal of developing therapeutics that enhance RCT to reduce the risk of cardiovascular disease. Many of the drugs and natural compounds examined target several transcription factors critical for ABCA1 expression in both macrophages and the liver. Likewise, several miRNAs target not only ABCA1 but also the same transcription factors that are critical for its high expression. However, after years of research and many preclinical and clinical trials, only a few leads have proven beneficial in this regard. In this review we discuss the various transcription factors that serve as drug targets for ABCA1 and provide an update on some important leads.

Endoplasmic reticulum stress: A common pharmacologic target of cardioprotective drugs.

Despite the advances made in cardiovascular disease prevention, there is still substantial residual risk of adverse cardiovascular events. Contemporary evidence suggests that additional reduction in cardiovascular disease risk can be achieved through amelioration of cellular stresses, notably inflammatory stress and endoplasmic reticulum (ER) stress. Only two clinical trials with anti-inflammatory agents have supported the role of inflammatory stress in cardiovascular risk. However, there are no clinical trials with selective ER stress modifiers to test the hypothesis that reducing ER stress can reduce cardiovascular disease. Nevertheless, the ER stress hypothesis is supported by recent pharmacologic studies revealing that currently available cardioprotective drugs share a common property of reducing ER stress. These drug classes include angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, mineralocorticoid receptor blockers, ß-adrenergic receptor blockers, statins, and select antiglycemic agents namely, metformin, glucagon like peptide 1 receptor agonists and sodium glucose cotransporter 2 inhibitors. Although these drugs ameliorate common risk factors for cardiovascular disease, such as hypertension, hypercholesterolemia and hyperglycemia, their cardioprotective effects may be partially independent of their principal effects on cardiovascular risk factors. Clinical trials with selective ER stress modifiers are needed to test the hypothesis that reducing ER stress can reduce cardiovascular disease.

The effect of black seed (Nigella sativa) extract on lipid metabolism in HepG2 cells.

Black seed extract stimulates apolipoprotein A-I (apo A-I) gene expression in hepatocytes and intestinal cells in part by elevating peroxisome proliferator-activated receptor α (PPARα) and retinoid X receptor α (RXRα) levels. To explore potential ramifications of these observations, we examined the effects of black seed extract on hepatocyte lipid content and expression of key transcriptional regulators of fatty acid ß-oxidation and lipogenesis in HepG2 cells. PPARα, peroxisome proliferator-activated receptor γ (PPARγ), RXRα, thyroid hormone receptor ß (TRß), sterol-responsive element binding protein 1 (SREBP1), and sterol-responsive element binding protein 2 (SREBP2) levels were measured in black seed extract treated liver-derived HepG2 cells. Black seed extract treatment increased PPARα and RXRα expression and decreased intracellular neutral lipid content. Black seed extract treatment increased TRß expression and activity, and PPARα activity. In contrast, PPARγ, SREBP1 and SREBP2 levels were decreased in black seed extract treated cells. Black seed extract treatment also increased acyl-CoA synthetase long chain family member 5 (ACSL5), peroxisomal acyl-CoA oxidase 1 (ACOX1), and carnitine palmitoyl transferase 1A (CPT-1A) expression, three PPARα-dependent rate-limiting genes that facilitate fatty acid oxidation, similar to fenofibrate. PPARα knockdown reversed the effects of fenofibrate and blackseed on ACSL5, ACOX1, and CPT-1A expression. In conclusion, black seed extract-mediated lipid lowering in HepG2 cells is associated with increased expression of fatty acid oxidation enzymes and PPARα and reduced lipogenic signaling. Thus black seed extract may be potentially beneficial in metabolic diseases such as diabetes, cardiovascular disease, and metabolic syndrome.

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.

Insulin mimetic effect of D-allulose on apolipoprotein A-I gene.

Several nutrients modulate the transcriptional activity of the apolipoprotein A-I (apo A-I) gene. To determine the influence of rare sugars on apo A-I expression in hepatic (HepG2) and intestinal derived (Caco-2) cell lines, apo A-I, albumin, and SP1 were quantified with enzyme immunoassay and Western blots while mRNA levels were quantified with real-time polymerase chain reaction. The promoter activity was measured using transient transfection assays with plasmids containing various segments and mutations in the promoter. D-allulose and D-tagatose, increased apo A-I concentration in culture media while D-sorbose and D-allose did not have any measurable effects. D-allulose did not increase apo A-I levels in Caco-2 cells. These changes paralleled the increased mRNA levels and promoter activity. D-allulose-response was mapped at the insulin response core element (IRCE). Mutation of the IRCE decreased the ability of D-allulose and insulin to activate the promoter. Treatment of HepG2 cells, but not Caco-2 cells, with D-alluose and insulin increased SP1 expression relative to control cells. D-allulose augmented the expression and IRCE binding of SP1, an essential transcription factor for the insulin on apo A-I promoter activity. D-allulose can modulate some insulin-responsive genes and may have anti-atherogenic properties, in part due to increasing apo A-I production. PRACTICAL APPLICATIONS: Coronary artery disease (CAD) is the number one cause of mortality in industrialized countries. A risk factor associated with CAD is low high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) concentrations in plasma. Thus, novel therapeutic agents or nutrients that upregulate apo A-I production should be identified. D-allulose and D-tagatose are used as sweeteners and may have favorable effects on insulin resistance and diabetes. This study shows that D-allulose and D-tagatose increases apo A-I production through increased transcription factor SP1-binding to insulin response element of the promoter. These sweeteners modulate some insulin responsive genes, increase the production of apo-A-I, and therefore may have anti-atherogenic properties.

Differential effects of cyclooxygenase-2 (COX-2) inhibitors on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells.

Selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib was pulled off the market because of its association with increased risk of adverse cardiovascular effects. The precise underlying mechanism for the differential effects of COX-2 inhibitors on cardiovascular risk is not known. Since endoplasmic reticulum (ER) stress is implicated in atherogenesis, we examined the effects of COX-2 inhibitors on ER stress in primary human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC), and human pulmonary artery endothelial cells (HPAEC). ER stress was measured in HCAEC treated with either tunicamycin (TM) or high-concentrations (27.5 mM) of dextrose (HD) using the secreted alkaline phosphatase (ES-TRAP) assay. Markers of the unfolded protein response (UPR) such as activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and phospho-PERK were measured by Western blot. Treatment of HCAEC with TM and HD decreased secreted alkaline phosphatase activity indicating increased ER stress. Treatment of cells exposed to TM or HD with celecoxib, meloxicam, ibuprofen, and acetylsalicylic acid, but not rofecoxib, resulted in a dose-dependent decrease in ER stress. High-dextrose and TM increased IRE1α and PERK phosphorylation and ATF6 and GRP78 expression. Treatment with celecoxib, but not rofecoxib, inhibited these markers of the UPR. Treatment with selective COX-2 inhibitors, with the exception of rofecoxib, suppressed ER stress as measured with both alkaline phosphatase activity assays and markers for the UPR. The inability of rofecoxib to inhibit ER stress, unlike the other cyclooxygenase inhibitors tested, may have contributed to its unfavorable effects on cardiovascular outcomes.

Inflammation, high-density lipoprotein and cardiovascular dysfunction.

PURPOSE OF REVIEW: This review describes the evidence that supports the hypothesis that high-density lipoprotein (HDL) is atheroprotective due to its antiinflammatory effects and benefits on vascular health. RECENT FINDINGS: Recent investigations have shown that HDL may inhibit atherosclerosis by promoting healthy endothelial function and by limiting or inhibiting the activation of macrophage and other immune cells. Receptors for HDL clearly regulate immune system function as well as cellular stress. Recent studies also suggest that participation of HDL in the process of reverse cholesterol transport may inhibit growth factor and cytokine receptor signaling by depleting cholesterol from lipid rafts. However, inflammation can also be associated with circulating dysfunctional HDL, which often possesses both prooxidative and proinflammatory properties. SUMMARY: These studies suggest that HDL-based therapeutics have potential in treating both acute and chronic conditions associated with inflammation. These studies also reveal several other pathways that may be targeted for therapeutic drug development.

Reduced cellular glucose transport confers natural protection against dextrose-induced superoxide generation and endoplasmic reticulum stress in domestic hen.

Normal blood glucose levels in avian species are two to fourfold higher than that in humans and the higher blood glucose levels in birds do not cause adverse effects. Endothelial cells isolated from the aorta of the domestic hen (Gallus gallus domesticus) and chicken aortic smooth muscle cells (CAOSMC) were compared to human coronary artery endothelial cells (HCAEC) and human primary aortic smooth muscle cells (HASMC). Superoxide (SO) generation was measured using a superoxide-reactive probe. ER stress was measured using the placental alkaline phosphatase assay (ES-TRAP). Glucose transport kinetics were determined using the 3 H-2-deoxyglucose tracer. Dextrose-induced SO generation and ER stress were significantly blunted in avian endothelial cells compared to human cells. The Vmax of glucose uptake (in nmoles/mg protein/min) in avian endothelial cells (0.0018 ± 0.0001) and smooth muscle cells (0.0015 ± 0.0007) was approximately 18-25 fold lower compared to the Vmax in HCAEC (0.033 ± 0.0025) and HASMC (0.038 ± 0.004) (all p < 0.0001). The Michaelis-Menten constant (Km) of transport was also significantly different (p < 0.0001) in avian species. The relative resistance of avian cells to dextrose-induced oxidative stress and ER stress is mostly the result of reduced cellular dextrose transport.

Effect of anti-hyperglycemic drugs on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells.

Endoplasmic reticulum (ER) stress plays a critical role in progression of diabetes and development of complications, notably cardiovascular disease. Some of the contemporary anti-hyperglycemic drugs have been shown to inhibit ER stress. To extend these observations, the effects of various anti-hyperglycemic agents were screened for their effects on ER stress. Seven classes of anti-hyperglycemic drugs were screened including sulfonylureas, meglitinides, metformin, α glucosidase inhibitors, thiazolidinedione, glucagon like peptide 1 (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT-2) inhibitors. ER stress was measured in human coronary artery endothelial cells (HCAEC) either treated with tunicamycin (TM) or cultured in hyperglycemic conditions (27.5 mM dextrose). The ER stress was measured with the secreted alkaline phosphatase (ES-TRAP) assay. Mediators of the unfolded protein response, including activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), phospho-inositol-requiring enzyme 1α (pIRE1α), IRE1α, phospho-protein kinase R (PKR)-like endoplasmic reticulum kinase (pPERK), and PERK were measured by Western blot. Metformin, GLP-1 receptor agonists (GLP-1, exendin 4, liraglutide, albiglutide, and lixisenatide) and SGLT-2 inhibitors (canagliflozin, dapagliflozin, and empagliflozin) were the only anti-hyperglycemic drugs screened that reduced ER stress caused by pharmacological (tunicamycin) or hyperglycemic conditions. High-dextrose and TM increased IRE1α and PERK phosphorylation and ATF6 and GRP78 expression, while treatment with metformin, liraglutide (a GLP-1 receptor agonist) and dapagliflozin (a SGLT-2 inhibitor), suppressed IRE1α and PERK phosphorylation as well as ATF6 and GRP78 expression. Thus, the cardioprotective effects of metformin, some of the GLP-1 receptor agonists and SGLT2 inhibitors may be partly related to their ability to reduce ER stress.