Unveiling the role of ABI3 and hub senescence-related genes in macrophage senescence for atherosclerotic plaque progression.

BACKGROUND: Atherosclerosis, characterized by abnormal arterial lipid deposition, is an age-dependent inflammatory disease and contributes to elevated morbidity and mortality. Senescent foamy macrophages are considered to be deleterious at all stages of atherosclerosis, while the underlying mechanisms remain largely unknown. In this study, we aimed to explore the senescence-related genes in macrophages diagnosis for atherosclerotic plaque progression. METHODS: The atherosclerosis-related datasets were retrieved from the Gene Expression Omnibus (GEO) database, and cellular senescence-associated genes were acquired from the CellAge database. R package Limma was used to screen out the differentially expressed senescence-related genes (DE-SRGs), and then three machine learning algorithms were applied to determine the hub DE-SRGs. Next, we established a nomogram model to further confirm the clinical significance of hub DE-SRGs. Finally, we validated the expression of hub SRG ABI3 by Sc-RNA seq analysis and explored the underlying mechanism of ABI3 in THP-1-derived macrophages and mouse atherosclerotic lesions. RESULTS: A total of 15 DE-SRGs were identified in macrophage-rich plaques, with five hub DE-SRGs (ABI3, CAV1, NINJ1, Nox4 and YAP1) were further screened using three machine learning algorithms. Subsequently, a nomogram predictive model confirmed the high validity of the five hub DE-SRGs for evaluating atherosclerotic plaque progression. Further, the ABI3 expression was upregulated in macrophages of advanced plaques and senescent THP-1-derived macrophages, which was consistent with the bioinformatics analysis. ABI3 knockdown abolished macrophage senescence, and the NF-κB signaling pathway contributed to ABI3-mediated macrophage senescence. CONCLUSION: We identified five cellular senescence-associated genes for atherogenesis progression and unveiled that ABI3 might promote macrophage senescence via activation of the NF-κB pathway in atherogenesis progression, which proposes new preventive and therapeutic strategies of senolytic agents for atherosclerosis.

LncRNA FPASL suppresses fibroblast proliferation through its DNA methylation via DNMT3b in hypertrophic scar.

Long noncoding RNAs (lncRNAs) are increasingly being implicated as key regulators of cell proliferation, apoptosis, and differentiation. However, the molecular mechanisms of specific lncRNAs in the context of hypertrophic scar remain largely unclear. Here, we find that the lncRNA FPASL (fibroblast proliferation-associated LncRNA) is downregulated in HS, and FPASL reduces fibroblast proliferation and colony formation and blocks cell cycle progression. Using GO annotation enrichment analysis along with AZC (a specific inhibitor of DNA methylation), we identify that DNA methylation is responsible for downregulating FPASL in hypertrophic scar. Subsequent studies demonstrate that high expression of DNMT3b inhibits FPASL expression in HS. Mechanistic study reveals a significant increase in fibroblast proliferation after transfection with LNA-FPASL, which is further inhibited by knockdown of DNMT3b. Thus, our study reveals that DNMT3b mediates hypermethylation of the lncRNA FPASL promoter and the downregulation of lncRNA FPASL promotes fibroblast proliferation in hypertrophic scar.

A novel lncRNA FPASL regulates fibroblast proliferation via the PI3K/AKT and MAPK signaling pathways in hypertrophic scar.

Hypertrophic scar is a problem for numerous patients, especially after burns, and is characterized by increased fibroblast proliferation and collagen deposition. Increasing evidence demonstrates that lncRNAs contribute to the development and progression of various diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly characterized. In this study, a novel fibroblast proliferation-associated lncRNA, named lncRNA FPASL (MSTRG.389905.1), which is mainly localized in the cytoplasm, is found to be downregulated in hypertrophic scar, as detected by lncRNA microarray and qRT-PCR. The full-length FPASL is characterized and further investigation confirms that it has no protein-coding potential. FPASL knockdown in fibroblasts triggers fibroblast proliferation, whereas overexpression of FPASL directly attenuates the proliferation of fibroblasts. Furthermore, target genes of the differentially expressed lncRNAs in hypertrophic scars and the matched adjacent normal tissues are enriched in fibroblast proliferation signaling pathways, including the PI3K/AKT and MAPK signaling pathways, as determined by GO annotation and KEGG enrichment analysis. We also demonstrate that knockdown of FPASL activates the PI3K/AKT and MAPK signaling pathways, and specific inhibitors of the PI3K/AKT and MAPK signaling pathways can reverse the proliferation of fibroblasts promoted by FPASL knockdown. Our findings contribute to a better understanding of the role of lncRNAs in hypertrophic scar and suggest that FPASL may act as a potential novel therapeutic target for hypertrophic scar.

Ischemic Postconditioning Protects against Aged Myocardial Ischemia/Reperfusion Injury by Transcriptional and Epigenetic Regulation of miR-181a-2-3p.

Ischemic postconditioning (IPostC) has been proposed as a strategy to mitigate the risk of ischemia/reperfusion (I/R) injury, and autophagy is involved in I/R-induced aged myocardial injury, while the underlying mechanism of IPostC-regulated autophagy is unknown. Here, we implemented miRNA sequencing analysis in aged cardiomyocytes to identify a novel miR-181a-2-3p after HPostC, which inhibits autophagy by targeting AMBRA1 in aged myocardium to protect I/R-induced aged myocardial injury. Mechanistically, we identified that IPostC can induce DNA hypomethylation and H3K14 hyperacetylation of miR-181a-2-3p promoter due to the decreased binding of DNMT3b and HDAC2 at its promoter, which contributes to enhancing the expression of miR-181a-2-3p. More importantly, cooperation of DNMT3b and HDAC2 inhibits the binding of c-Myc at the miR-181a-2-3p promoter in aged cardiomyocytes. In summary, IPostC attenuates I/R-induced aged myocardial injury through upregulating miR-181a-2-3p expression, which is an attribute to transcriptional and epigenetic regulation of its promoter. Our data indicate that miR-181a-2-3p may be a potential therapeutic target against I/R injury in aged myocardium.

miR-30a-5p promotes glomerular podocyte apoptosis via DNMT1-mediated hypermethylation under hyperhomocysteinemia.

Abnormal elevation of homocysteine (Hcy) level is closely related to the development and progression of chronic kidney disease (CKD), with the molecular mechanisms that are not fully elucidated. Given the demonstration that miR-30a-5p is specifically expressed in glomerular podocytes, in the present study we aimed to investigate the role and potential underlying mechanism of miR-30a-5p in glomerular podocyte apoptosis induced by Hcy. We found that elevated Hcy downregulates miR-30a-5p expression in the mice and Hcy-treated podocytes, and miR-30a-5p directly targets the 3'-untranslated region (3'-UTR) of the forkhead box A1 (FOXA1) and overexpression of miR-30a-5p inhibits FOXA1 expression. By nMS-PCR and MassARRAY quantitative methylation analysis, we showed the increased DNA methylation level of miR-30a-5p promoter both and . Meanwhile, dual-luciferase reporter assay showed that the region between --1400 and --921 bp of miR-30a-5p promoter is a possible regulatory element for its transcription. Mechanistic studies indicated that DNA methyltransferase enzyme 1 (DNMT1) is the key regulator of miR-30a-5p, which in turn enhances miR-30a-5p promoter methylation level and thereby inhibits its expression. Taken together, our results revealed that epigenetic modification of miR-30a-5p is involved in glomerular podocyte injury induced by Hcy, providing a diagnostic marker candidate and novel therapeutic target in CKD induced by Hcy.

miR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications.

Atherosclerosis is a serious age-related disease, which has a tremendous impact on health care globally. Macrophage inflammation is crucial for the initiation and progression of atherosclerosis, and microRNAs (miRNAs) recently have emerged as potent modulators of inflammation, while the underlying mechanisms of its involvement in homocysteine (Hcy)-mediated macrophage inflammation of atherosclerosis remain largely unknown. Here, we demonstrated that elevated Hcy inhibits the expression of miR-195-3p, which in turn enhances IL-31 expression and thereby causes the secretion of macrophages pro-inflammatory factors IL-1ß, IL-6 and TNF-α and accelerate atherosclerosis. Furthermore, we identified that Hcy can induce DNA hypermethylation and H3K9 deacetylation of miR-195-3p promoter due to the increased the binding of DNMT3a and HDAC11 at its promoter. More importantly, Sp1 interacts with DNMT3a suppressed the binding of HDAC11 at miR-195-3p promoter and promoted its transcription. In summary, our results revealed a novel mechanism that transcriptional and epigenetic regulation of miR-195-3p inhibits macrophage inflammation through targeting IL-31, which provides a candidate diagnostic marker and novel therapeutic target in cardiovascular diseases induced by Hcy.

SNF5 promotes IL-1ß expression via H3K4me1 in atherosclerosis induced by homocysteine.

Homocysteine (Hcy) is a strong and independent risk factor of atherosclerosis. It can accelerate atherosclerosis through increased production of inflammatory factors, especially interleukin-1 ß (IL-1ß), while the precise mechanisms remain to be well elucidated. In this study, we investigated the role of the tumor suppressor gene SNF5 related to switch/sucrose non-fermentable complex (SWI/SNF) in the occurrence and development of atherosclerosis induced by Hcy. Using Hyperhomocysteinemia (HHcy) atherosclerotic model with apolipoprotein E knockout (ApoE-/-) mice fed with high-methionine diet, we showed that Hcy aggravates inflammation in macrophages during the atherosclerotic plaque formation. Further analysis showed that SNF5 promotes IL-1ß expression and secretion. In addition, due to the existence of H3K4 methylation signals in the vicinity of IL-1ß, we found that Hcy significantly promotes the expression of H3K4me1, and lysine-specific histone demethylase 1A (KDM1A) acts as a transcriptional repressor to regulate the expression of H3K4me1 by demethylating H3K4me1. In summary, our results demonstrated that Hcy up-regulates the expression of SNF5 through KDM1A, resulting in an increased level of H3K4me1 modification and IL-1ß in macrophages, which in turn promotes the formation of atherosclerosis. Our study will provide more evidence for further revealing the specific mechanism of Hcy-induced inflammation and the diagnosis, prevention, and treatment of atherosclerosis.

Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to SCARB1 promoter. Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at least partly accelerated Hcy-mediated atherosclerosis through promoting lipid accumulation in foam cells, which was attributed to the decreased binding of SP1 to SCARB1 promoter. In our point, these findings will provide novel insight into an epigenetic mechanism for atherosclerosis.

Hypermethylation of endoplasmic reticulum disulfide oxidase 1α leads to trophoblast cell apoptosis through endoplasmic reticulum stress in preeclampsia.

Abnormal trophoblast cell apoptosis is implicated in the pathogenesis of pregnancy-related disorders including preeclampsia (PE), and endoplasmic reticulum (ER) stress has been considered as a novel pathway in the regulation of cell apoptosis. In this study, we observed that both apoptosis and ER stress are triggered in trophoblast cells under hypoxia as well as in the placenta of PE rats. Quantitative polymerase chain reaction and Western blot analysis showed that the expression of endoplasmic reticulum disulfide oxidase 1α (ERO1α) is suppressed in trophoblast cells under hypoxia due to the hypermethylation of the ERO1α promoter region, and the inhibition of ERO1α expression plays an important role in ER stress and trophoblast cell apoptosis. Furthermore, we found that DNA methyltransferase 1 (DNMT1) is a key methyltransferase for DNA methylation in the regulation of ERO1α expression, and the binding level of DNMT1 to the ERO1α promoter is markedly elevated under hypoxia although DNMT1 expression is inhibited by hypoxia, suggesting that the binding level of DNMT1 to the ERO1α promoter region rather than the DNMT1 expression level contributes to the hypermethylation of ERO1α. Taken together, these results demonstrate that the hypermethylation of ERO1α mediated by increased binding of DNMT1 to the ERO1α promoter leads to trophoblast cell apoptosis through ER stress in the placenta of PE rats, which shed insight into the etiology of PE and might present a validated therapeutic target for the treatment of PE.

Homocysteine-induced extracellular superoxide dismutase and its epigenetic mechanisms in monocytes.

Although a modest homocysteine (Hcy) elevation is associated with an increased cardiovascular risk, the underlying mechanisms whereby Hcy triggers the accumulation of cholesterol and the roles of the extracellular superoxide dismutase (EC-SOD) in the development of foam cells have not yet been elucidated. In this study, we found both increased numbers of foam cells and an accumulation of cholesterol, and the H(2)O(2) and oxidized low-density lipoprotein content also increased. Levels of EC-SOD were significantly suppressed by Hcy, however, while 5-azacytidine (AZC), a potent DNA methyltransferase (DNMT) inhibitor, increased the expression of EC-SOD. A quantitative real-time PCR of EC-SOD revealed that Hcy (100 micromol l(-1)) accelerates DNA methylation of EC-SOD, but selectively increases the activity of DNA methyl transferase 1 (DNMT1). It showed that Hcy can reduce binding of methyl CpG and binding protein 2 (MeCP2) but has no effect on the activity of DNMT3. Moreover, chromatin immunoprecipitation assays demonstrated that Hcy increased the binding of acetylated histone H3 and H4 in monocytes. Based on the fact that the binding of MeCP2 with the EC-SOD was completely suppressed by AZC and trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor], it is indicated that DNA methylation and HDAC mediate the binding of MeCP2 with EC-SOD gene. In conclusion, the study found that Hcy accelerates the development of foam cells by repressing EC-SOD transcription, and that Hcy exerts this function by upregulating DNA methylation via suppression of HDAC activity and increased DNMT1 activity.

[Effects of peroxisome proliferators activated receptors on caveolin-1 expression in foam cells].

OBJECTIVE: To study the effect of peroxisome proliferators activated receptors (PPAR) alpha, gamma ligand on ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 expressions and cholesterol, ox-LDL contents in human monocyte derived foam cells. METHOD: Malondialdehyde (MDA) was measured by TBARS method, ox-LDL detected by ELISA method, cholesterol measured by fluorescence spectrophotometric method, ABCA1, caveolin-1 mRNA and protein expressions determined by RT-PCR and Western blot, in human monocytes, foam cells [human monocyte-derived macrophage induced by myristate acetate (PMA) further treated with 50 mg/L ox-LDL for 24 h], foam cells plus 10 micromol/L pioglitazone for 48 h, foam cells plus 5 micromol/L clofibrate for 48 h. RESULT: The intracellular total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE), ox-LDL and lipid peroxide were significantly increased and the membrane expressions of ABCA1, caveolin-1 were down-regulated in foam cells compared to monocytes (all P < 0.05) and these changes were significantly attenuated by cotreatment with PPARalpha, gamma ligand. CONCLUSION: The anti-atherosclerosis effects of PPARalpha, gamma ligand are related to reducing cholesterol contents and up-regulating ABCA1, caveolin-1 expressions in foam cells.

Hyperhomocysteinemia-mediated DNA hypomethylation and its potential epigenetic role in rats.

Hyperhomocysteinemia (HHcy), which is an independent risk factor for atherosclerosis, might cause dysregulation of gene expression, but the characteristics and key links involved in its pathogenic mechanisms are still poorly understood. The objective of the present study was to investigate the effect of HHcy on DNA methylation and the underlying mechanism of homocysteine (Hcy)-induced DNA methylation. HHcy was induced in Sprague-Dawley rats after 4 weeks of a low, medium or high methionine diet. The levels of total homocysteine, S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) were detected by high-performance liquid chromatography. The expression levels of genes and proteins of S-adenosylhomocysteine hydrolase, DNA methyltransferase and methyl-CpG-binding domain 2 were detected by real-time reverse transcription-polymerase chain reaction and Western blot analysis. A high-throughput quantitative methylation assay using fluorescence-based real-time polymerase chain reaction was employed to determine the levels of DNA methylation. The results indicated that HHcy induced the elevation of AdoHcy concentration, the decline of AdoMet concentration, the ratios of AdoMet/AdoHcy and the RNA and protein expression of S-adenosylhomocysteine hydrolase and methyl-CpG-binding domain 2, as well as an increase of DNA methyltransferase activity. With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer CCGG sequences to CpG islands. Increasing levels of HHcy significantly increased genome hypomethylation in B1 repetitive elements. The impacts of different levels of HHcy showed that the varied detrimental effects of HHcy could be attributed to different concentrations through different mechanisms. In mild and moderate HHcy, the Hcy might primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism. However, at high Hcy concentrations, the impacts might be more injurious through oxidative stress, apoptosis and inflammation.

Ligands of peroxisome proliferator-activated receptor inhibit homocysteine-induced DNA methylation of inducible nitric oxide synthase gene.

Homocysteine (Hcy) is a risk factor for atherosclerosis. It is generally accepted that inducible nitric oxide synthase (iNOS) is a key enzyme in the regulation of vascular disease. The aim of the present study is to investigate the effects of peroxisome proliferator-activated receptor ligands on iNOS in the presence of Hcy in human monocytes. Foam cells, induced by oxidize low density lipoprotein (ox-LDL) and phorbol myristate acetate (PMA) in the presence of different concentrations of Hcy, clofibrate and pioglitazone in human monocytes for 4 d, were examined by oil red O staining. The activity of iNOS was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. The capability of DNA methylation was measured by assaying endogenous C5 DNA methyltransferase (C5MTase) activity, and the iNOS promoter methylation level was determined by quantitative MethyLight assays. The results indicated that Hcy increased the activity of C5MTase and the level of iNOS gene DNA methylation, resulting in a decrease of iNOS expression. Clofibrate and pioglitazone could antagonize the hcy effect on iNOS expression through DNA methylation, resulting in attenuation of iNOS transcription. These findings suggested that Hcy decreased the expression of iNOS by elevating iNOS DNA methylation levels, which can repress the transcription of some genes. Peroxisome proliferator-activated receptor alpha/gamma ligands can down-regulate iNOS DNA methylation, and could be useful for preventing Hcy-induced atherosclerosis by repressing iNOS expression.