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. 2021 Apr 23;117(5):1339-1357.
doi: 10.1093/cvr/cvaa219.

NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli

Suvi Linna-Kuosmanen  1 , Vanesa Tomas Bosch  1 , Pierre R Moreau  1 , Maria Bouvy-Liivrand  2 , Henri Niskanen  1 , Emilia Kansanen  1 , Annukka Kivelä  1 , Juha Hartikainen  2   3 , Mikko Hippeläinen  3 , Hannu Kokki  2   4 , Pasi Tavi  1 , Anna-Liisa Levonen  1 , Minna U Kaikkonen  1
Affiliations

NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli

Suvi Linna-Kuosmanen et al. Cardiovasc Res. .

Abstract

Aims: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs.

Methods and results: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease.

Conclusion: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.

Keywords: miR-21-5p • miR-100-5p •  NRF2 • Atherosclerosis.

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Figures

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Graphical abstract
Figure 1
Figure 1
Global characterization of NRF2-regulated transcriptional mechanisms in human vascular endothelial cells. (A) Motif enrichment in up-regulated and down-regulated transcripts from the GRO-seq data in HUVECs, HAECs, HASMCs, and CD14+ macrophages under oxPAPC stimuli. (B) Ingenuity pathway upstream regulator analysis of differentially expressed genes (FDR < 0.05) in HUVECs after OXPAPC stimulation, generated with IPA (QIAGEN). (C) Percentage of down-regulated (black) and up-regulated (red) genes found in the vicinity (2 or 50 kb) of NRF2 peaks detected by ChIP-Seq in HAECs (GSM2394418) under OxPAPC condition. (D) Reads distribution among TSS and gene body of up- and down-regulated genes proximal to NRF2 ChIP-Seq peaks (GSM2394418) under oxPAPC and normoxia.
Figure 2
Figure 2
Identification of NRF2-regulated miRNome in vascular cells. (A) Global expression of HUVEC and HAEC miRNAs, those found in both, and putative and confirmed NRF2-regulated miRNAs. (B) Percentages of NRF2-regulated miRNAs of the total miRNA expression in HUVECs and HAECs. For all: n = 2, mean ± SD. Genomic loci of mir-22 (C) and mir-106b/25/93 cluster (D). Histone and chromatin segmentation data are ENCODE data from UCSC Genome Browser. Chromatin segmentation track shows promoters in red, enhancers in orange, and active chromatin regions in green. AREs are determined using previously published tool. HiC interactions were visualized with WashU Epigenome Browser.
Figure 3
Figure 3
Most NRF2-regulated miRNAs have similar expression patterns in other vascular cell types. GRO-seq signals for mir-21 (A), mir-29a/29b-1 (B), and mir-125b-1/let-7a-2/miR-100 (C) loci are shown. TVs mark the different transcript variants as determined in Ref. Heatmap shows transcript variant expression based on GRO-seq data for the listed miRNAs.
Figure 4
Figure 4
NRF2-regulated miRNAs involved in atherosclerosis-related functions. (A) IPA’s comparison analysis for HUVECs and HAECs showing the changes in atherosclerosis-related functions. (B) IPA’s VEGF pathway from HUVEC data. Stars mark the molecules directly affected by miRNAs. Red marks up-regulation and cyan down-regulation. (C) Overview of the observed changes in atherosclerosis context.
Figure 5
Figure 5
Detailed analysis of NRF2-regulated miR-21-5p and miR-100-5p expressions. Genomic loci of mir-21 (A) and mir-125b-1/let-7a-2/mir-100 cluster (B). Histone and chromatin segmentation data are ENCODE data from UCSC Genome Browser. Chromatin segmentation track shows promoters in red, enhancers in orange, and active chromatin regions in green. AREs are determined using previously published tool. HiC interactions were visualized with WashU Epigenome Browser. (C) miR-21-5p expression in HUVECs under oxPAPC stimuli for indicated times compared to control samples. (D) miR-100-5p expression in HUVECs under oxPAPC stimuli for indicated times compared to control samples, and extracellular miRNA expression measured from the growth media of oxPAPC-treated cells and from the exosomes extracted from the growth media compared to control samples. (E) miRNA expression in KEAP1 and NRF2 overexpressing cells. (n = 3, mean ± SD, unpaired two-tailed t-test, ****P < 0.0001, **P < 0.01, *P < 0.05).
Figure 6
Figure 6
miR-21-5p and miR-100-5p mediate oxPAPC effects on VEGFA/MYC pathway. (A) miRNA-target network from miRNet., (B) Illustration of VEGF/MYC pathway. (C) Effects of miR-21-5p overexpression and miR-100-5p inhibition on MTOR, HIF1A, VEGFA, and MYC expressions. (n = 3, mean ± SD, unpaired two-tailed t-test, ****P < 0.0001, ***P < 0.001).
Figure 7
Figure 7
miR-21-5p and miR-100-5p response during senescence and in coronary artery disease patients. (A) miRNA expression in passage (p) 8–16 cells compared to p4 cells. (B) miRNA levels in growth medium of p8–p16 cells compared to p4 cells. (C) miRNA levels in exosomes extracted from the growth medium of p8–p16 cells compared to p4 cells. (D) As in (A) but under oxPAPC stimuli. (E) As in (B) but under oxPAPC stimuli. (F) As in (C) but under oxPAPC stimuli. For (A–F) figures: n = 3, mean ± SD, unpaired two-tailed t-test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. (G) miR-21-5p and miR-100-5p levels in human pericardial fluid of coronary artery disease (CAD) patients (n = 22, mean ± SD). (H) miRNA levels in pericardial fluid exosomes of control and CAD groups (n = 16 for control, n = 22 for CAD, mean ± SD, unpaired two-tailed t-test, *P < 0.05). (I) miRNA levels in pericardial fluid exosomes of CAD patients divided according to the severity of heart failure symptoms (NYHA classes I–IV), where I is no symptoms and IV severe symptoms (n = 4–6 for NYHA II, n = 5 for NYHA III and n = 6–8 for NYHA IV, mean ± SD).
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References

    1. Gimbrone MA, García-Cardeña G.. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 2016;118:620–636. - PMC - PubMed
    1. Bochkov VN, Oskolkova OV, Birukov KG, Levonen A-L, Binder CJ, Stöckl J.. Generation and biological activities of oxidized phospholipids. Antioxid Redox Signal 2010;12:1009–1059. - PMC - PubMed
    1. Lee S, Birukov KG, Romanoski CE, Springstead JR, Lusis AJ, Berliner JA.. Role of phospholipid oxidation products in atherosclerosis. Circ Res 2012;111:778–799. - PMC - PubMed
    1. Andreou I, Sun X, Stone PH, Edelman ER, Feinberg MW.. miRNAs in atherosclerotic plaque initiation, progression, and rupture. Trends Mol Med 2015;21:307–318. - PMC - PubMed
    1. Nazari-Jahantigh M, Egea V, Schober A, Weber C.. MicroRNA-specific regulatory mechanisms in atherosclerosis. J Mol Cell Cardiol 2015;89:35–41. - PubMed

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