Endothelial Progenitor Cells Senescence Induced By Remnant-like Lipoproteins Via The Oxidative Stress Mechanism And The Screening Of MicroRNA Profile

BackgroundCoronary heart disease(CHD) is one of the highest human morbidity and mortality diseases, and the initial and crucial step of which is atherosclerosis. Hypertension, diabetes and smoking have been demonstrated to be three classical risk factors for atherosclerosis. In the area of atherosclerosis caused by dyslipidemia, Emerging epidemiological and clinical studies have been proceeded to determined the atherogenic features of low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C).RLPs (Remnant-like lipoproteins), the lipolytic products of triglyceride-rich lipoproteins including very low-density lipoprotein and chylomicron, are smaller in size, contain less triglyceride, more cholesterol esters and Apo-E than their precursors, and therefore have more atherogenic potentials in theory. It is argued that human lie in postprandial state in the most of the day(beyond 12h). the concentration of the plasma RLPs are significantly elevated in the postprandial state when compared with those in the fasting state. Elevated level of RLPs cholesterol is considered to be a coronary risk factor and a predictor of cardiovascular events independent of HDL-C and LDL-C in healthy subjects and patients with coronary artery disease (CAD). Further investigations demonstrated that RLPs were closely related to endothelial dysfunction. Several atherosclerotic properties of RLPs such as pro-inflammatory, contributing to the formation of foam cells and increasing the level of oxidative stress, have been determined. However, the exact mechanism by which endothelial dysfunction caused by RLPs remains entirely unclear.Until now, there are just a few studies about the association between RLPs and EPCs senescence which is worth to further determined. Oxidative stress hypothesis is one of the mechanisms for the onset and process of atherosclerosis. Like ox-LDL, whether RLPs accelerate EPCs senescence or not should be elucidated in the future investigation.As a consequence of semi-conservative DNA replication, the extreme termini of chromosomes are not duplicated completely, resulting in successive telomeres shortening with each cell division. Critical telomere shortening or telomere dysfunction contributes to replicative senescence, which is so-called telomere mechanism. Telomerase, can catalyze the addition of telomeric repeats (TTAGGG)n to telomeres and stabilize telomeres length, delay or prevent from cellular senescence. It has been demonstrated that statin, vascular endothelial growth factor(VEGF), HDL and estrogen can activate the PI3K/Akt signal pathway, decrease the level of intracellular oxidative stress and increase telomerase activity, and then delay or block cellular senescence. Can RLPs accelerate EPCs senescence via the above telomere mechanism?MicroRNAs (miRNAs) can bind to messenger RNA (mRNA) targets, generally within their 3 untrans-lated region, and regulate gene expression by catalyzing the cleavage of mRNA in the post-transcriptional level or repressing mRNA translation. Recent studies have demonstrated that miRNAs were closely associated with intracellular oxidative stress and then regulate cellular proliferation and senescence. Recent study have determined that miR-34a overexpression can contribute to the onset of EPCs senescence through inhibiting silent information regulator 1 (SIF-1). Can RLPs lead to acceleration of EPCs senescence via alteration of some miRNAs profile? Can the taget genes of the senescence-associated miRNAs be predicted? To date, the relative studies are scarce and is deserved to be further explored.ObjectiveIn this present study, EPCs were treated with RLPs, and two senescence-associated markers?-Gal and p16INK4a were detected to determine whether RLPs contribute to the onset of EPCs. Next, After pretreatment with atorvastatin(Ator) superoxide dismutase (SOD), the protein levels of FAK, Akt and their phosphorylation were measured to evaluate the effect of RLPs on EPCs senescence via the above reactive oxigen species-dependent pathway; to futher explore the relationship between the above ROS-dependent pathway and telomere pathway, and finally the senescence-associated miRNAs and their target genes were filtered and predicted. This present study aimed to elucidate the exact mechanism by which RLPs lead to acceleration of EPCs senescence, and provide the theoretical basis for atherosclerosis.Methods1. The high-fat meal model was enacted. The peripheral blood obtained from volunteers was used to isolate RLPs by the combinations of immunoaffinity, dialyzation and ultracentrifugation methods, etc.2. RLPs were identified by the use of the combination of enzyme detection, Brawald protein detection and agarose electrophoresis analysis.3. Peripheral blood mononuclear cells were isolated by the density gradient centrifugation and differenciated into EPCs which were identified by flow cytometry analysis.4. EPCs were incubated with a series of concentrations of RLPs, the protein level of p16INK4a was detected by western blot analysis. The proximal concentration of RLPs was determined at 100?g protein/mL. EPCs were divided into four groups:control, RLPs, RLPs+Ator, RLPs+SOD. The number of senescent cells was measured by?-gal staining analysis. 5. Nitrotyrosine, which represents Peroxynitrite (ONOO—) generation and reflects the level of reactive oxygen species, was detected by Immunofluorescence method.6. EPCs were incubated with a series of concentrations of RLPs. Then telomerase activity was measured by TRAP-ELISA analysis, and the optimal concentration of RLPs was confirmed as 100?g protein/mL7. The protein level of hTERT was detected by the combination of western blot and Immunoprecipitation (IP); the levels of the total protein hTERT?FAK, Akt and their phosphorylation were evaluated by western blot analysis.8. The miRNAs profiles in control and senescent EPCs induced by RLPs(100?g protein/mL) were analyzed by the microarray method, and were confirmed by the real time RT-PCR method.9. The change of the transcripts in the non-RLPs-treated and RLPs-treated EPCs was evaluated by use of the two-color Whole Human Genome 4×44k oligo microarrays, in combination with bioinformatics algorithms, commonly predicted the target genes of those senescence-associated miRNAs.Results1. RLPs were isolated successfully from peripheral blood by the combinations of immunoaffinity, dialyzation and ultracentrifugation methods, etc. EPCs were isolated and cultured by the the density gradient centrifugation and differenciation methods.2. RLPs dose-dependently contributed to an increase in the protein expression of p16INK4a. When compared with control, RLPs(100?g protein/mL) led to a significant increase in the number of senescence-associated?-gal-positive cells(P< 0.01). The pretreatment with Ator(1?mol/L) or SOD(50U/mL) reversed the above inhibitory effects of RLPs(P< 0.01).3. When compared with control, RLPs(100?g protein/mL) remarkably upregulated nitrotyrosine production, and pretreatments with Ator(1?mol/L) or SOD(50U/mL) significantly inhibited nitrotyrosine generation.4. RLPs inhibited telomerase activity in a dose-dependent manner with a maximal inhibitory effect achieved at 100?g protein/mL, and meanwhile downregulated the protein level of hTERT and its phosphorylation. Pretreatment with Ator and SOD significantly upregulated the decreased telomerase activity and hTERT expression in RLPs-incubated EPCs.5. RLPs (100?g protein/mL) significantly inhibited the protein expressions of FAK,Akt and their phosphorylation. Likewise, Pretreatment with Ator (1?mol/L) and SOD (50U/mL) improved the above inhibitory effects of RLPs. 6. The microarray result indicated that the expression profile of senescent EPCs induced by RLPs(100?g protein/mL) is that of 723 human miRNAs,16 were upregulated while 6 were down-regulated when compared with control. Of those 21 different miRNAs, miR-148b, miR-155 and miR-513c were significantly upregulated while miR-574-3p was significantly downregulated in senescent cells. In accordance with the microarray result, the result from real time RT-PCR analysis showed that the alterations of three miR-148b, miR-155 and miR-574-3p reached significant difference.7. Potential target genes of miR-148b and miR155 that were predicted by the combined approach of these algorithms miRanda, PicTar, MirTarget, and the Whole Human Genome 4×44 k oligo microarray:miR-148b:CCKBR, ACVR1, DNMT1,ITGA5,GAP43; miR-155:BACH1,FBXO11.Conclusions1. RLPs can result in acceleration of EPCs senescence, which may be closely related to increased generation of intrcellular reactive oxygen species.2. RLPs may be inhibit the reactive oxygen species-dependent pathway (FAK/PI3K/Akt signal pathway), inactivate telomerase activity, and then accelerate the onset of EPCs senescence. 3. RLPs can result in the alteration of senescent-associated miRNAs and their target genes, indicating that miRNAs may take part in regulating RLP-induced EPCs senescence.