Gabriela M. Sanda

MiR-486 and miR-92a Identified in Circulating HDL Discriminate between Stable and Vulnerable Coronary Artery Disease Patients

Small non-coding microRNAs (miRNAs) are implicated in gene regulation, including those involved in coronary artery disease (CAD). Our aim was to identify whether specific serum miRNAs present in the circulating lipoproteins (Lp) are associated with stable or vulnerable CAD patients. A cardiovascular disease-focused screening array was used to assess miRNAs distribution in sera collected from 95 CAD patients: 30 with stable angina (SA), 39 with unstable angina (UA), 26 at one month after myocardial infarction (MI) and 16 healthy control subjects. We found that miR-486, miR-92a and miR-122 presented the highest expression in CAD sera. These miRNA together with miR-125a, miR-146a and miR-33a were further individually analyzed by TaqMan assays. The results were consistent with PCR-array screening data that all of these miRNAs were significantly increased in CAD patients compared to controls. Using a binary logistic regression model, we established that miR-486 and miR-92a in association with some high-density lipoprotein (HDL) components can designate vulnerable CAD patients. Further, all classes of Lp were isolated from sera by density gradient ultracentrifugation. Analysis of the selected miRNAs in each Lp class showed that they were associated mainly with HDL, miR-486 and miR-92a having the highest levels. In UA and MI patients, miR-486 prevailed in HDL2, while miR-92a prevailed in HDL3, and their levels discriminate between stable and vulnerable CAD patients. We identified two circulating miRNAs that in association with some lipid metabolism biomarkers can be used as an additional tool to designate vulnerable CAD patients.

Anti-oxidant and anti-inflammatory mechanisms of amlodipine action to improve endothelial cell dysfunction induced by irreversibly glycated LDL.

Amlodipine, alone or in combination with other drugs, was successfully used to treat hypertension. Our aim was to evaluate the potential of amlodipine (Am) to restore endothelial dysfunction induced by irreversibly glycated low density lipoproteins (AGE-LDL), an in vitro model mimicking the diabetic condition. Human endothelial cells (HEC) from EA.hy926 line were incubated with AGE-LDL in the presence/absence of Am and the oxidative and inflammatory status of the cells was evaluated along with the p38 MAPK and NF-κB signalling pathways. The cellular NADPH activity, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine levels in the culture medium and the adhesion of human monocytes to HEC were measured by chemiluminescence, UHPLC, Western Blot and spectrofluorimetric techniques. The gene expression of NADPH subunits (p22(phox), NOX4), eNOS and inflammatory molecules (MCP-1, VCAM-1) were determined by Real Time PCR, while the protein expression of p22(phox), MCP-1, iNOS, phospho-p38 MAPK and phospho-p65 NF-κB subunit were measured by Western Blot. Results showed that in HEC incubated with AGE-LDL, Am led to: (i) decrease of the oxidative stress: by reducing p22(phox), NOX4, iNOS expression, NADPH oxidase activity, 4-HNE and 3-nitrotyrosine levels; (ii) decrease of the inflammatory stress: by the reduction of MCP-1 and VCAM-1 expression, as well as of the number of monocytes adhered to HEC; (iii) inhibition of ROS-sensitive signalling pathways: by decreasing phosphorylation of p38 MAPK and p65 NF-κB subunits. In conclusion, the reported data demonstrate that amlodipine may improve endothelial dysfunction in diabetes through anti-oxidant and anti-inflammatory mechanisms.

Probiotics determine hypolipidemic and antioxidant effects in hyperlipidemic hamsters

SCOPE Hyperlipidemia, hyperglycemia, and the oxidative stress are among the known risk factors of atherosclerosis. Our aim was to assess the hypolipidemic and antioxidant effects of a probiotic mix (Lactobacillus acidophilus and Bifidobacterium animalis) in hyperlipidemic hamsters (HL). METHODS AND RESULTS Male Golden Syrian hamsters developed hyperlipidemia after 21 weeks of fat diet. For the last 5 weeks of experiment, ten HL were treated with the probiotic mix (HLP), ten received water (HL). Ten animals received standard chow (N). Increase of plasma total cholesterol (TC), triglycerides (TG), phospholipids (PL), oxidized LDL, glucose, of 4-hydroxynonenal (4-HNE) in plasma, liver, and myocardium, and of intestinal Niemann Pick C1 like 1 (NPC1L1) and microsomal TG transfer protein (MTTP) expression was observed in HL versus N. The probiotic mix decreased plasma TC, TG, PL, oxidized LDL, 4-HNE, and glucose levels and increased paraoxonase-1 activity, decreased NPC1L1 and MTTP protein expression compared to HL. In HLP liver, a significant reduction of TC, TG, and fatty acids was observed. PL increased and 4-HNE levels decreased in the liver and myocardium of HLP versus HL. CONCLUSION Our data support the administration of probiotics to humans because of their hypolipidemic (through decreasing intestinal NPC1L1 and MTTP) and antioxidant effects (stimulating HDL-associated paraoxonase-1).

Analysis of circulating microRNAs that are specifically increased in hyperlipidemic and/or hyperglycemic sera.

MicroRNAs (miRNAs) are small non-coding RNA sequences that regulate gene expression post-transcriptionally by translation inhibition or mRNA degradation. The aim of the present study was to analyze serum miRNAs modulated by hyperlipidemia and/or hyperglycemia and to correlate them with biochemical parameters within lipid metabolism. Five selected circulating miRNAs (miR-125a-5p, miR-146a, miR-10a, miR-21 and miR-33a) were individually analyzed by TaqMan miRNA assays along with lipid and inflammation parameters in sera from 20 hyperlipidemic (HL) and/or hyperglycemic (HG) patients, and compared with data from five normolipidemic/normoglycemic subjects. Results showed: (1) the levels of all the analyzed circulating miRNA were increased in HL sera and correlated positively with sera’s lipid and inflammatory parameters; (2) circulating miR-125a-5p and miR-146a levels were increased in HG and/or HL sera; (3) all selected miRNAs were detected in α-lipoprotein fraction from sera, and miR-33a was also present in β-lipoprotein fraction; (4) miRNA concentrations were increased in the α-lipoprotein fraction from HL sera. These data show a statistically significant correlation of the analyzed miRNA with increased lipids, specifically with α- and β-lipoproteins, and CRP and IL-1β levels in HL and/or HG sera, suggesting a contribution of these miRNAs to the atherosclerotic process.

HDL inhibit endoplasmic reticulum stress by stimulating apoE and CETP secretion from lipid-loaded macrophages

The role of HDL in the modulation of endoplasmic reticulum (ER) stress in macrophage-derived foam cells is not completely understood. Therefore, we aimed to investigate whether HDL may inhibit ER stress in correlation with the secretion of apoE and CETP from lipid-loaded macrophages. To this purpose, THP-1 macrophages were loaded with lipids by incubation with human oxidized LDL (oxLDL) and then exposed to human HDL3. ER stress signaling markers, protein kinase/Jun-amino-terminal kinase (SAPK/JNK p54/p46) and eukaryotic initiation factor-2α (eIF2α), as well as the secreted apoE and CETP, were evaluated by immunoblot analysis. Out of the many different bioactive lipids of oxLDL, we tested the effect of 9-hydroxy-octadecadienoic acid (9-HODE) and 4-hydroxynonenal (4-HNE) on ER stress. Tunicamycin was used as positive control for ER stress induction. Results showed that oxLDL, 9-HODE and 4-HNE induce ER stress in human macrophages by activation of eIF-2α and SAPK/JNK (p54/p46) signaling pathways. OxLDL stimulated apoE and CETP secretion, while tunicamycin determined a reduction of the secreted apoE and CETP, both in control and lipid-loaded macrophages. The addition of HDL3 to the culture medium of tunicamycin-treated cells induced: (i) the reduction of ER stress, expressed as decreased levels of eIF-2α and SAPK/JNK, and (ii) a partial recovery of the secreted apoE and CETP levels in lipid-loaded macrophages. These data suggest a new mechanism by which HDL3 diminish ER stress and stimulate cholesterol efflux from lipid-loaded macrophages.

Hyperlipidemia‐induced hepatic and small intestine ER stress and decreased paraoxonase 1 expression and activity is associated with HDL dysfunction in Syrian hamsters

SCOPE We aimed at investigating the mechanisms linking hyperlipidemia (HL) with dysfunctional HDL and its main antioxidant enzyme, paraoxonase1 (PON1). PON1 expression and activity was determined in the small intestine, liver, and sera of normal and HL hamsters and associated with the ER stress (ERS) and the development of aortic valve lesions. METHODS AND RESULTS Male Golden Syrian hamsters were fed standard chow (N) or standard diet with 3% cholesterol and 15% butter for 16 weeks. All hamsters on fat diet developed HL, 50% also hyperglycemia (HLHG) and a fourfold increased homeostasis model assessment of insuline resistance. PON1 expression was reduced in the small intestine and liver (N > HL > HLHG) along with the increased extent of ERS, oxidized lipids, and decreased expression of liver X receptors beta (LXRβ) in the small intestine, peroxisome proliferator-activated receptor-γ (PPARγ) in the liver, and of the glucose transporter 4 in the myocardium. Serum PON1 levels decreased along with the increase of oxidized LDL and lesion areas of the aortic valves (N > HL > HLHG). CONCLUSION The fat diet activates the ERS and oxidative stress, decreases LXRβ, PPARγ, and PON1 in the small intestine, liver, and sera of all HL animals, in parallel with the appearance of atherosclerotic lesions in the aortic valves.

Apolipoprotein A–I stimulates cholesteryl ester transfer protein and apolipoprotein E secretion from lipid-loaded macrophages; the role of NF-κB and PKA signaling pathways

Cholesteryl ester transfer protein (CETP) and apolipoprotein E (apoE) are secreted by macrophages. Apolipoprotein A-I (apoA-I) is a potent inducer of apoE secretion from lipid-loaded macrophages, but its effect on CETP is not known. We aimed to identify the signaling pathways involved in apoA-I and HDL-mediated regulation of CETP and apoE secretion from lipid-loaded macrophages. THP-1 macrophages were loaded with lipids by incubation with human copper-oxidized LDL. The cells were subsequently exposed to human purified apoA-I or HDL(3) with/without inhibitors of NF-κB (TPCK) or PKA (H89). CETP and apoE in the cultured cells and media were quantified by real-time PCR and Western blot. Results showed that in lipid-loaded macrophages: (i) CETP and apoE gene expression and secretion were increased in the presence of apoA-I, and further increased by inhibition of NF-kB with TPCK; (ii) CETP and apoE gene expression and secretion were reduced by the inhibition of PKA with H89; (iii) PKA-gamma subunit was activated by oxidized LDL and moreover by apoA-I. We also showed that: (i) siRNA-mediated CETP gene silencing diminished apoE secretion from both non-loaded and lipid-loaded macrophages; (ii) addition of apoA-I partially restored apoE secretion from lipid-loaded macrophages with the silenced CETP gene. In conclusion, our data suggest a new mechanism by which apoA-I stimulates CETP secretion, in addition to apoE, from lipid loaded macrophages, a process involving NF-κB inhibition and/or PKA pathway activation.

Hyperglycemia Determines Increased Specific MicroRNAs Levels in Sera and HDL of Acute Coronary Syndrome Patients and Stimulates MicroRNAs Production in Human Macrophages.

We aimed to determine the levels of microRNAs (miRNAs) in sera and HDL of acute coronary syndrome (ACS) compared to stable angina (SA) patients with/without hyperglycemia, and evaluate comparatively the functional effect of these sera on the processing machinery proteins (Drosha, DGCR8, Dicer) and miRNAs production in human macrophages. MiRNAs levels in sera and HDL from 35 SA and 72 ACS patients and 30 healthy subjects were measured by using microRNA TaqMan assays. MiR-223, miR-92a, miR-486, miR-122, miR-125a and miR-146a levels were higher in the hyperglycemic ACS compared to normoglycemic sera. MiR-223 and miR-486 prevailed in HDL2, while miR-92a predominated in HDL3, all three miRNAs discriminating between ACS and SA patients; their levels were increased in HDL from hyperglycemic ACS patients versus normoglycemic ones. The incubation of human macrophages with sera from ACS and SA patients showed that all patients’ sera induced an increase of Drosha, DGCR8 and Dicer expressions and of selected miRNAs levels compared to control sera, the effect being higher in the case of hyperglycemic versus normoglycemic ACS sera. The addition of glucose to SA and ACS sera increased Drosha, DGCR8 and Dicer expression and miRNAs levels in the exposed macrophages. In conclusion, hyperglycemia is associated with increased miR-223, miR-92a, miR-486 levels in HDL, which discriminate between ACS and SA patients. Exposure of human macrophages to ACS compared to SA sera determines the upregulation of Drosha, DGCR8 and Dicer expression and the increase of selected miRNAs production, the effect being augmented by an increased glucose concentration.

Dysfunctional high-density lipoproteins have distinct composition, diminished anti-inflammatory potential and discriminate acute coronary syndrome from stable coronary artery disease patients

There is a stringent need to find means for risk stratification of coronary artery diseases (CAD) patients. We aimed at identifying alterations of plasma high-density lipoproteins (HDL) components and their validation as dysfunctional HDL that could discriminate between acute coronary syndrome (ACS) and stable angina (SA) patients. HDL2 and HDL3 were isolated from CAD patients’ plasma and healthy subjects. ApolipoproteinAI (apoAI), apoAII, apoCIII, malondialdehyde (MDA), myeloperoxidase (MPO), ceruloplasmin and paraoxonase1 (PON1) were assessed. The anti-inflammatory potential of HDL subfractions was tested by evaluating the secreted inflammatory molecules of tumor necrosis factor α-activated endothelial cells (EC) upon co-incubation with HDL2 or HDL3. We found in ACS versus SA patients: 40% increased MPO, MDA, apoCIII in HDL2 and HDL3, 35% augmented apoAII in HDL2, and in HDL3 increased ceruloplasmin, decreased apoAII (40%) and PON1 protein and activity (15% and 25%). Co-incubation of activated EC with HDL2 or HDL3 from CAD patients induced significantly increased levels of secreted inflammatory molecules, 15–20% more for ACS versus SA. In conclusion, the assessed panel of markers correlates with the reduced anti-inflammatory potential of HDL subfractions isolated from ACS and SA patients (mostly for HDL3 from ACS) and can discriminate between these two groups of CAD patients.

Oxidized LDL-Exposed Human Macrophages Display Increased MMP-9 Expression and Secretion Mediated by Endoplasmic Reticulum Stress.

Oxidatively modified low‐density lipoproteins (oxLDL) alter the proper function of the endoplasmic reticulum (ER), inducing ER stress (ERS), which consequently activates inflammatory pathways in macrophages. Matrix metalloproteinase‐9 (MMP‐9) is the main protease acting on the degradation of the extracellular matrix and the ensuing destabilization of the atherosclerotic plaque. We aimed to investigate whether ERS induced by oxLDL or tunicamycin (TM) in human macrophages is associated with the stimulation of MMP‐9 expression and secretion. The results showed that oxLDL induced in THP‐1 macrophages: (i) increase of MMP‐9 gene expression and its pro‐form secretion, (ii) intracellular accumulation of 7‐ketocholesterol, (iii) ERS activation (increased eIF2α phosphorylation, XBP1 and CHOP mRNA levels, and Grp78 protein expression), and (iv) oxidative stress (increased levels of reactive oxygen species and NADPH oxidase activity). Incubation of macrophages with ERS inducer, TM determined the secretion of both pro‐ and active‐form of MMP‐9 and oxidative stress. Treatment of oxLDL or TM‐incubated cells with ERS inhibitor, sodium phenylbutyrate decreased MMP‐9 gene expression, secretion, and activity. The inhibitor of NADPH oxidase, apocynin, decreased XBP‐1 and CHOP mRNA levels, and MMP‐9 gene expression and secretion in oxLDL‐exposed cells. In conclusion, oxLDL stimulate MMP‐9 expression and secretion in human macrophages by mechanisms involving ERS. J. Cell. Biochem. 118: 661–669, 2017.