Obstructive Sleep Apnea-induced Endothelial Dysfunction Is Mediated by miR-210

Abstract

Rationale: Obstructive sleep apnea (OSA)-induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea-hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced [Formula: see text]o₂ rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element-binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron-sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia-increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae.

Keywords: endothelium; miR-210; mitochondrial dysfunction; obstructive sleep apnea.

Figure 1.

Endothelial cell–derived microRNA 210 (miR-210) is induced in obstructive sleep apnea (OSA). (A and B) miR expression concentrations were mined from the online data set GSE116909 (Gene Expression Omnibus) (35). This data set was collected from experiments using human umbilical vein endothelial cells (HUVECs) exposed to sustained (0.9% O₂) or intermittent (0.9% O₂ for 1 h and then normoxia for 36 min per cycle) hypoxia for the indicated time or cycle (A). Expression of miR-210-3p is highlighted by red dots and lines (B). (C and D) miR-210 expression in HUVECs exposed to sustained or intermittent hypoxia for the indicated time (C) or cycles (D) detected using quantitative PCR (qPCR); U6 was an internal control. (E) Circulating concentration of miR-210 was quantified using qPCR in age- and sex-matched healthy control subjects (HCs; n = 17 in cohort 1, n = 22 in cohort 2) and individuals with OSA (n = 47 in cohort 1, n = 18 in cohort 2) in two different cohorts. The apnea–hypopnea index (AHI) was evaluated using the sleep apnea test. (F) Pearson correlation analysis of correlation between log₂ FC of miR-210 serum concentration and AHI. (G) CD31⁺ MPs were isolated from serum from patients with OSA (n = 13) and age-matched HCs (n = 14). The concentration of miR-210 was detected using qPCR; Caenorhabditis elegans miR-39 was a spike-in control. Data are shown as mean ± SEM. Statistical significance was determined using a two-tailed t test or the Mann-Whitney U test. *P < 0.05 compared with control or HCs. FC = fold change; miR = microRNA; MP = microparticles.

Figure 2.

Mitochondrial function impaired by SREBP2 (sterol regulatory element–binding protein 2)–microRNA 210 (miR-210) in endothelial cells (ECs) revealed by RNA sequencing (RNA-seq). (A) Human umbilical vein ECs were treated with pooled obstructive sleep apnea (OSA) serum (from three patients with OSA with apnea–hypopnea index > 30) or age- and sex-matched pooled healthy control (HC) serum for 24 hours or transfected with miR-210 or scramble control for 48 hours. RNA was extracted from these cells for RNA-seq. Differentially expressed genes (DEGs [log₂ fold change > 1.5; P < 0.05]) were compared between OSA/HC RNA-seq and miR-210/control RNA-seq. We used Metascape Gene Ontology enrichment analysis of the overlapped DEGs from OSA/HC and miR-210/control RNA-seq results plotted as −log₁₀(P value). (B) The heatmap shows the RNA concentrations of mitochondrial function–related genes from OSA/HC and miR-210/control RNA-seq. ACKR3 = atypical chemokine receptor 3; ARL2 = ADP ribosylation factor like GTPase 2; BSG = basigin (Ok blood group); CHCHD1 = coiled-coil-helix-coiled-coil-helix domain containing 1; COX14 = cytochrome C oxidase assembly factor COX14; COX4I1 = cytochrome C oxidase subunit 4I1; Ctrl = control; CYBA = cytochrome B-245 alpha chain; DDIT3 = DNA damage inducible transcript 3; DEG = differentially expressed gene; DPP7 = dipeptidyl peptidase 7; FIS1 = fission, mitochondrial 1; FKBP8 = FKBP prolyl isomerase 8; FOSL1 = FOS like 1, AP-1 transcription factor subunit; HRAS = HRas proto-oncogene, GTPase; HSPA5 = heat shock protein family A (Hsp70) member 5; IFI27 = interferon alpha inducible protein 27; MRPL23 = mitochondrial ribosomal protein L23; NDUF = NADH:ubiquinone oxidoreductase; PRDX5 = peroxiredoxin 5; SEC61A1 = SEC61 translocon subunit alpha 1; SNN = stannin; SOD1 = superoxide dismutase 1; TGF = transforming growth factor; TIMM13 = translocase of inner mitochondrial membrane 13; TNF = tumor necrosis factor; TUSC2 = tumor suppressor 2, mitochondrial calcium regulator; UQCRQ = ubiquinol-cytochrome C reductase complex III subunit VII.

Figure 3.

Obstructive sleep apnea (OSA) serum–induced microRNA 210 (miR-210) impairs mitochondrial function in endothelial cells (ECs). (A–D) Human umbilical vein ECs were transfected with pro–miR-210 or scramble Ctrl for 48 hours (A and D) or transfected with anti–miR-210 for 24 hours, followed by incubation of pooled OSA or healthy control (HC) serum for another 24 hours (A–D). In A, representative confocal images of mitochondrial morphology are shown. Mitochondria were visualized by using TOM20 antibody (50 cells counted for each replicate). Scale bar, 2.5 μm. Tubular: most mitochondria in ECs were >10 μm long; intermediate: mitochondria were <≈10 μm; fragment: most mitochondria were spherical (no clear length or width). In B, mitochondrial abundance was assessed using MT. In C, mitochondrial membrane potential was detected using JC-1 staining. In B and C, scale bar, 200 μm. In D, the OCR was detected using Seahorse (https://www.agilent.com/en/promotions/seahorse-xf-hs-mini-analyzer?gclid=Cj0KCQiAmaibBhCAA​RIsAKUlaKRfws3HpgQdtLNaDZzm9zOaQ6XoyUJTszxz-VYXPn6NArHA5PqxBPwaArPxEALw_wcB&gclsrc=aw.ds) assay. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Bonferroni post hoc test or two-tailed Student’s t test. *P < 0.05 compared with Ctrl or HC or between indicated two groups. AA/R = antimycin A and rotenone; Ctrl = control; FCCP = carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; FITC = fluorescein isothiocyanate; Max Resp = maximal respiration; MT = MitoTracker; OCR = $\dot{\text{V}}$o₂ rate; Pro = production; Resp Cap = respiratory capacity; TRITC = tetramethylrhodamine.

Figure 4.

Obstructive sleep apnea (OSA) serum–induced miR-210 inhibits iron–sulfur cluster assembly enzyme (ISCU) in ECs. (A and B) Human umbilical vein endothelial cells (HUVECs) were treated with 10% pooled OSA or healthy control (HC) serum for 24 hours. The mRNA concentrations of indicated genes (A) or ISCU protein (B) were detected using quantitative PCR (qPCR) or western blot analysis, respectively. β-Actin was used as an internal control. (C and D) HUVECs were transfected with 20 or 30 nM microRNA 210 (miR-210) (C) or with anti–miR-210 combined with OSA or HC serum (D). ISCU mRNA concentration was detected using qPCR. (E) HUVECs were treated with pooled OSA or HC serum for 48 hours, followed by Ago2 (argonaute RISC catalytic component 2) RNA immunoprecipitation. Ago2-associated miR-210 and ISCU mRNA concentrations were quantified using qPCR. (F) Bovine aorta ECs were transfected with Luc-ISCU-3′UTR (WT) or Luc-ISCU-3′UTR (MT) for 24 hours, then pooled OSA or HC serum for another 24 hours. Luc activity was measured with Renilla as transfection control. miR-210 was quantified using qPCR. Data are shown as mean ± SEM from at least three independent experiments. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons or the two-tailed Mann-Whitney U test. *P < 0.05 compared with control, HC, or indicated groups. Ctrl = control; EC = endothelial cell; Luc = luciferase; MAO = monoamine oxidase; MnSOD = manganese superoxide dismutase; MT = miR-210 targeting site TCCCTCT replaced by ACGCACA; NRF1 = nuclear respiratory factor 1; TFAM = transcription factor A, mitochondrial; UCP2 = uncoupling protein 2; UTR = untranslated region; WT = wild-type.

Figure 5.

SREBP2 (sterol regulatory element–binding protein 2) transactivates miR-210 in ECs. (A) The putative transcription factors binding to the microRNA 210 (miR-210) promoter were predicted using TRANSFAC; transcription factors are listed in order of binding scores. The sequences of common binding motifs in the miR-210 active promoter and SREBP2 binding motif were deduced. (B) Epigenetic landscapes of assay for transposase-accessible chromatin (ATAC) signals (light blue) and H3K27ac (acetylated lysine 27 of histone H3) enrichment (red) in the promoter region of miR-210 in human umbilical vein endothelial cells (HUVECs); the highlighted region was defined as miR-210 active promoter according to these epigenetic landscapes. Putative sterol regulatory elements (SREs) and SREBP2 overexpression-induced ATAC signals (purple) in the miR-210 promoter are shown. HUVECs were infected with Ad-null or Ad-SREBP2 for 24 hours. SREBP2 binding to the miR-210 promoter was detected by SREBP2 chromatin immunoprecipitation (ChIP) followed by ChIP-quantitative PCR (qPCR). The qPCR primers are depicted by number of SRE sites. (C) HUVECs were treated with 10% pooled OSA or HC serum for 24 hours. SREBP2 protein concentration was detected using western blot with α-tubulin as a loading control. (D) RNA was isolated from lung and serum from EC-specific SREBP2-overexpression transgenic mice and their sex- and age-matched WT littermates. (E and F) HUVECs were transfected with si-SREBP2 (E) or treated with betulin (0.2 mg/ml; F) for 24 hours, followed by incubation with 10% OSA or HC serum for an additional 24 hours. In D–F, miR-210 concentration was detected using qPCR. Data are shown as mean ± SEM from at least three independent experiments. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons or the two-tailed Mann-Whitney U test. *P < 0.05 compared with control or between indicated groups. Ad = adenovirus; BMI1 = BMI1 proto-oncogene, polycomb ring finger; BP2-Tg = endothelial cell–specific SREBP2-overexpression transgenic mice; EC = endothelial cell; FC = fold change; GATA3 = GATA binding protein 3; HSF1 = heat shock transcription factor 1; IKZF2 = IKAROS family zinc finger 2; KLF = KLF transcription factor; LMO2 = LIM domain only 2; MAFB = MAF BZIP transcription factor B; PAX8 = paired box 8; SNAI1 = Snail family transcriptional repressor 1; SREBPF = sterol regulatory element–binding transcription factor; TF = transcription factor; WT = wild-type; ZBTB7A = zinc finger and BTB domain containing 7A; ZEB2 = zinc finger E-box binding homeobox 2.

Figure 6.

Obstructive sleep apnea (OSA) serum decreases iron–sulfur cluster assembly enzyme (ISCU) via SREBP2 (sterol regulatory element–binding protein 2) induction of microRNA 210 (miR-210) in endothelial cells (ECs). (A, B, and D) Human umbilical vein ECs were transfected with SREBP2 siRNA (si-BP2) or pretreated with betulin (0.2 mg/ml) for 24 hours, then HC or OSA serum for another 24 hours. (C and E) ECs were isolated from aorta of EC-specific SREBP2-overexpression transgenic mice (three males and three females) and their WT littermates (three males and three females). Protein concentrations of SREBP2, ISCU, and β-actin (loading control) were detected using western blot analysis (A and B). SREBP2 and ISCU mRNA concentrations were detected using quantitative PCR with β-actin as an internal control (A–C). Mitochondrial respiratory complex I activity was evaluated using enzyme activity assay (D). Data are shown as mean ± SEM from at least three independent experiments. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons or the two-tailed Mann-Whitney U test. *P < 0.05 compared with control or between two indicated groups. BP2-Tg = endothelial cell–specific SREBP2-overexpression transgenic; HC = healthy control; MT DNA = mitochondrial DNA; WT = wild-type.

Figure 7.

SREBP2 (sterol regulatory element–binding protein 2)–microRNA 210 (miR-210) impairs mitochondrial function in mice exposed to intermittent hypoxia (IH). (A–G) Eight-week-old C57Bl/6j mice were exposed to normoxia (21% O₂; control [Ctrl], n = 10 male mice) or IH (cycles of 60 s 21% O₂ + 30 s 10% O₂ for 8 h/d), with (n = 12 males) or without (n = 15 males) betulin. (A and G) Body weight, SBP, and DBP were assessed before and 2 weeks after IH treatment or under normoxia. The concentrations of miR-210 in circulation (B) and in CD31⁺ MPs (C) were quantified using quantitative PCR. Caenorhabditis elegans microRNA 39 was used as a spike-in control. SREBP2 and ISCU1/2 (iron–sulfur cluster assembly enzyme 1/2) mRNA concentration (D) or protein concentrations (E and H) in mice aortas were quantified using quantitative PCR or western blot, respectively. (F) Lung tissues were extracted from mice, and mitochondrial respiratory complex I activity was assessed. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by the Bonferroni post hoc test or two-tailed Student’s t test (for parametric data). Statistical significance of nonparametric data was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons or the two-tailed Mann-Whitney U test. *P < 0.05 between indicated groups. CD31 = cluster of differentiation 31; DBP = diastolic blood pressure; FC = fold change; MP = microparticles; SBP = systolic blood pressure.