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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Regulation of apolipoprotein A-I gene expression by the histamine H1 receptor: Requirement for NF-κB.

AIMS: Earlier it had been found by us that apolipoprotein A-I (apo A-I) is suppressed by histamine in HepG2 cells. Histamine has been shown to regulate NF-κB activity, though not in hepatocytes. Therefore we examined the role of the histamine receptors and NF-κB in histamine-mediated apo A-I gene expression in HepG2 liver cells. MAIN METHODS: The effect of histamine on histamine H1 receptor expression, and NF-κB p65 and p50 subunits was examined by Western blot. Histamine H1 receptor involvement was examined by loss-of-function (via siRNA) and gain-of-function studies overexpressing the histamine H1 receptor. The requirement for the p65 subunit of NF-κB for histamines effect was elucidated by loss-of-function studies (siRNA). Finally, the effect of histamine on NF-κB binding to the apo A-I gene promoter was examined by chromatin immunoprecipitation. KEY FINDINGS: Treatment of HepG2 cells with histamine had no effect on histamine H1 receptor expression. However, treatment with histamine increased NF-κB p65 and p50 subunit expression significantly. At low levels, the exogenous histamine H1 receptor plasmid suppressed apo A-I gene promoter activity while addition of higher levels of plasmid DNA actually increased apo A-I gene promoter activity. Inhibition of NF-κB activity with SN50 prevented histamine from repressing apo A-I promoter activity as did silencing p65 expression via siRNA. Finally, treatment with histamine increased binding of the p65 subunit of NF-κB to the apo A-I gene promoter. SIGNIFICANCE: Histamine suppresses apo A-I gene expression in hepatocytes via the histamine H1 receptor by elevating NF-κB expression and binding to the apo A-I promoter.

Inhibition of hepatic apolipoprotein A-I gene expression by histamine.

In a recent high throughput analysis to identify drugs that alter hepatic apolipoprotein A-I (apo A-I) expression, histamine receptor one (H1) antagonists emerged as potential apo A-1 inducing drugs. Thus the present study was undertaken to identify some of the underlying molecular mechanisms of the effect of antihistaminic drugs on apo AI production. Apo A-I levels were measured by enzyme immunoassay and Western blots. Apo A-I mRNA levels were measured by reverse transcription real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the internal control. The effects of histamine and antihistamines on apo A-I gene were determined by transient transfection of plasmids containing the apo A-I gene promoter. Histamine repressed while (H1) receptor antagonist azelastine increased apo A-I protein and mRNA levels within 48 h in a dose-dependent manner. Azelastine and histamine increased and suppressed, respectively, apo A-I gene promoter activity through a peroxisome proliferator activated receptor α response element. Treatment of HepG2 cells with other H1 receptor antagonists including fexofenadine, cetirizine, and diphenhydramine increased apo A-I levels in a dose-dependent manner while treatment with H2 receptor antagonists including cimetidine, famotidine, and ranitidine had no effect. We conclude that H1 receptor signaling is a novel pathway of apo A1 gene expression and therefore could be an important therapeutic target for enhancing de-novo apo A-1 synthesis.

High-Throughput Analysis Identifying Drugs That Regulate Apolipoprotein A-I Synthesis.

Apolipoprotein A-I (apo A-I) is the primary antiatherogenic protein in high-density lipoprotein (HDL). Despite the controversy as to the clinical effectiveness of raising HDL, the search is ongoing for safe and effective drugs that increase HDL and apo A-I levels. To identify novel compounds that can increase hepatic apo A-I production, two drug libraries were screened. The NIH clinical collection (NCC) and the NIH clinical collection 2 (NCC2) were purchased from Evotec (San Francisco, CA). The NCC library contains 446 compounds and the NCC2 library contains 281 compounds, all dissolved in dimethylsulfoxide at a concentration of 10 mM. Hepatoma-derived cells (HepG2) and primary hepatocytes in culture were treated with various compounds for 24 h and apo A-I in media samples was measured by enzyme immunoassay. Samples with significant changes in apo A-I concentrations were retested in independent experiments by Western blot analysis to confirm the immunoassay findings. Of a total of 727 compounds screened at a concentration of 50 µM, 15 compounds increased hepatic apo A-I production by 35%-54%, and 9 compounds lowered hepatic apo A-I concentrations in the culture media by 25%-52%. Future trials should explore the clinical effectiveness of these agents when standard doses of these drugs are used in humans.

Inhibition of endoplasmic reticulum stress and oxidative stress by vitamin D in endothelial cells.

Endoplasmic reticulum (ER) stress and oxidative stress promote endothelial dysfunction and atherosclerosis. Since vitamin D has been shown in several studies to lower the risk of cardiovascular disease, we examined the effects of vitamin D on ER stress and oxidative stress in endothelial cells. ER stress was measured using the placental secreted alkaline phosphatase assay and oxidative stress was measured by hydroethidine fluorescence. Expression of ER stress markers, including glucose-regulated protein 78, c-jun N-terminal kinase 1 phosphorylation, and eukaryotic initiation factor 2α phosphorylation, as well as X-box binding protein-1 splicing were measured in tunicamycin (TM)-treated human umbilical endothelial cells (HUVEC) treated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and other vitamin D analogs. When TM and 1,25-(OH)2D3 were added simultaneously, 1,25-(OH)2D3 prevented ER stress. However, the effect was much stronger when cells were pre-treated with 1,25-(OH)2D3 for 24-h. However, ER stress was not inhibited by 25-OH vitamin D3 (25-OHD3) or the vitamin D analog EB1089. Both ZK191784 and the vitamin D metabolite 24,25-dihydroxyvitamin D3 were as effective as 1,25-(OH)2D3 in preventing ER stress. Similar effects were observed dextrose-induced stress. All of the compounds tested, except for 25-OHD3, inhibited dextrose-induced (27.5mM) oxidative stress and ER stress. Although TM with and without 1,25-(OH)2D3 had no effect on VDR expression, inhibition of VDR expression via siRNA prevented 1,25-(OH)2D3, ZK191784, EB1089, and 24,25-dihydroxyvitamin D3 from inhibiting dextrose-mediated SO generation. Furthermore, each vitamin D analog, with the exception of 25-OHD3, prevented dextrose-induced toxicity. These results suggest that vitamin D has a protective effect on vascular endothelial cells.

Angiotensin II receptor one (AT1) mediates dextrose induced endoplasmic reticulum stress and superoxide production in human coronary artery endothelial cells.

BACKGROUND: Renin-angiotensin-aldosterone system (RAAS) has been implicated in diabetes-related vascular complications partly through oxidative stress. OBJECTIVE: To determine the role of angiotensin II receptor subtype one (AT1) in dextrose induced endoplasmic reticulum (ER) stress, another cellular stress implicated in vascular disease. METHODS: Human coronary artery endothelial cells with or without AT1 receptor knock down were treated with 27.5mM dextrose for 24h in the presence of various pharmacologic blockers of RAAS and ER stress and superoxide (SO) production were measured. Transfection of cells with AT1 antisense RNA knocked down cellular AT1 by approximately 80%. The ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay and western blot analysis of glucose regulated protein 78 (GRP78), c-jun-N-terminal kinase 1 (JNK1), phospho-JNK1, eukaryotic translation initiation factor 2α (eIF2α) and phospho-eIF2α measurements. 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. RESULTS: In cells with AT1 knock down, dextrose induced ER stress was significantly blunted and treatment with 27.5mM dextrose resulted in significantly smaller increase in SO production compared to 27.5mM dextrose treated and sham transfected cells. Dextrose induced ER stress was reduced with pharmacologic blockers of AT1 (losartan and candesartan) and mineralocorticoid receptor blocker (spironolactone) but not with angiotensin converting enzyme inhibitors (captopril and lisinopril). The dextrose induced SO generation was inhibited by all pharmacologic blockers of RAAS tested. CONCLUSIONS: The results indicate that dextrose induced ER stress and SO production in endothelial cells are mediated at least partly through AT1 receptor activation.

Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells.

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1-3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1ß, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.