Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction

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  Figure 1.. Generation of an endothelium-specific mouse model to study the function of miR-217 in vivo. A, miR-217 expression is associated with endothelial aging. qPCR analysis of miR-217 expression in aortas of young (6–9 wk old) and aged (2 y old) C57/Bl6 mice. B, Constructs used to generate the endothelium-specific miR-217 mouse model. In ROSA26miR217KI mice, the miR-217 precursor construct is inserted within the Rosa26 locus, where it is preceded by a LoxP-flanked transcriptional stop site. ROSA26miR217KI mice were crossed with endothelium-specific and tamoxifen-inducible VE-Cad (VE-cadherin) Cre ERT2TG mice to generate miR-217KI mice. C, Immunohistochemical detection of GFP expression in CD31+ aortic endothelial cells of VE-Cad CreERT2 control mice (miR-217+/+ VE-Cad CreERT2TG/+) and miR-217–overexpressing miR-217KI mice (miR-217KI/+ VE-Cad CreERT2TG/+) after tamoxifen (TMX) injection. D, qPCR analysis of aortic miR-217 and Sirt1 (silent information regulator 1) expression in control and endothelium-specific miR-217–overexpressing miR-217KI mice 12 wk post-TMX injection. Each dot represents an individual mouse. CD indicates cluster of differentiation; GFP, green fluorescent protein;…

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  Figure 2.. miR-217 expression impairs endothelial physiology. A, Acetylcholine (ACh)-induced NO production in aortas from male control apoE−/− mice (miR-217+/+ VE-Cad CreERT2TG/+ apoE−/−) and miR-217KI apoE−/− mice (miR-217KI/+ VE-Cad CreERT2TG/+ apoE−/−) fed with HFD for 12 wk. B, Concentration-response curves to ACh or the NO donor diethylamine NONOate (DEA-NO) and maximal relaxation to ACh of aorta from male control apoE−/− mice and miR-217KI apoE−/− mice. C, Concentration-response curve to phenylephrine (Phe) of mesenteric resistance arteries, from male control apoE−/− mice and miR-217KI apoE−/− mice. *P<0.05 vs control apoE−/− mice by 2-way ANOVA followed by Bonferroni post-test. Maximal relaxation was analyzed by the Mann-Whitney U test. D, Echocardiography assessment of systolic (S) and diastolic (D) aortic arch lumen diameter (top) and right carotid lumen diameter (bottom) in control and miR-217 apoE KO (knockout) mice fed with HFD for 12 wk. E, Systolic and diastolic blood pressure (BP) in control apoE−/− and miR-217KI apoE−/− mice fed with HFD for 10 to 11 wk. F, BP and carotid diameters in control apoE−/− and miR-217KI apoE−/− mice fed an HFD before and 1 wk after resveratrol…

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  Figure 3.. Endothelial miR-217 expression promotes atherosclerosis and coronary artery disease. A and B, Quantification of atherosclerotic lesions by red oil staining of aortic sinus and proximal aorta heart sections in control apoE−/− mice (miR-217+/+ VE-Cad CreERT2TG/+ apoE−/−, n=12) and miR-217KI apoE−/− mice (miR-217 KI/+ VE-Cad CreERT2TG/+ apoE−/−, n=9) fed an HFD for 7 wk. A, Representative red oil–stained aortic sinus cross sections from control apoE−/− and miR-217KI apoE−/− HFD-fed mice (left) and quantification of atherosclerotic lesion area through the aortic sinus and ascending aorta (right). Each data point represents mean lesion area at the specified distance from the aortic sinus. B, Total atherosclerotic lesion volume in aortic sinus of control apoE−/− and miR-217KI apoE−/− mice, determined by calculating the area under the curve for each mouse from lesion area graphs (mm2; Figure II in the Data Supplement). C, Macrophage infiltration area quantified by Mac2 immunofluorescence in atherosclerotic lesions from control apoE−/− and miR-217KI apoE−/− HFD-fed mice. Each data point represents total Mac2+ area in the aortic sinus cross section of an individual mouse. D, Necrotic core…

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  Figure 4.. miR-217 expression promotes left ventricular dysfunction. A, Left ventricular diastolic function assessed as the ratio of Doppler-detected peak early diastolic velocity (E′) to peak late diastolic velocity (A′) in control apoE−/− mice (miR-217+/+ VE-Cad CreERT2TG/+ apoE−/−) and miR-217KI apoE−/− mice (miR-217KI/+ VE-Cad CreERT2TG/+ apoE−/−) fed an HFD for 8 wk. Right, Representative echocardiography images. B, D2 pulmonary vein velocity in control apoE−/− and miR-217KI apoE−/− mice fed an HFD for 8 wk. C, Left ventricular ejection fraction in control apoE−/− and miR-217KI apoE−/− mice determined by echocardiography after 10 wk on an HFD. Data are from 2 independent experiments. Each dot represents an individual mouse. *P<0.05, **P<0.01 by unpaired t test (A–C). apoE indicates apolipoprotein E; LVID;d, left ventricular internal diameter in diastole; and LVID;s, left ventricular internal diameter in systole.

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  Figure 5.. Inhibition of endogenous miR-217 improves vascular contractile function and reduces atherosclerosis development. A, miR-217-Sponge (miR-217SPG) construct used for vascular miR-217 inhibition. Four miR-217 complementary sites, separated by 4-nt spacers, were placed downstream of GFP in the pHRSIN lentiviral vector. B, CD31 and GFP immunostaining in ascending aorta sections of apoE−/− mice inoculated through the jugular vein with control or miR-217SPG pHRSIN lentivirus. C, Concentration-response curves to acetylcholine (left) and the NO donor diethylamine NONOate (DEA-NO; right) and (D) effect of L-NAME on the concentration-response curve to phenylephrine (Phe), in aortic segments from male control and miR-217SPG–infected apoE−/− mice fed HFD for 12 wk. E, Red oil staining analysis of atherosclerotic lesion area in aortic sinus and ascending aorta sections from control and miR-217SPG–infected apoE−/− mice fed an HFD for 12 wk. Left, Representative red oil–stained cross sections 320 µm from the aortic sinus in control and miR-217SPG–infected mice. *P<0.05 by unpaired t test or ANOVA. 3'UTR indicates the three prime untranslated region; Ach, acetylcholine; apoE, apolipoprotein E; CD,…

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  Figure 6.. Effect of miR-217 on endothelial transcriptome. Study of gene networks regulated by miR-217 in primary mouse endothelial cells. A, Pathway activity prediction in significantly changed Cardiovascular Development and Functions analysis of RNA sequencing (RNA-Seq) gene expression quantification in endothelial cells isolated from lungs (MLECs) of miR-217KI apoE−/− or control apoE−/− mice. Pathway activity prediction is shown with a color code (blue, inhibited; orange, activated). B, Circus plot showing the fold change expression (logFC; left) of genes of significantly changed cardiovascular-related pathways (1–5; right). Gene expression was quantified by RNA-Seq in MLECs derived from miR-217KI apoE−/− or control apoE−/− mice. C, Ingenuity canonical pathway analysis from significantly changed pathways grouped in the 5 general cardiovascular-related categories shown in B. Pathway activity prediction is shown with a color code (blue, inhibited; orange, activated). P values were corrected for multiple testing using the Benjamini-Hochberg (B–H) false discovery rate. D, qPCR analysis of gene expression in control apoE−/− and miR-217KI apoE−/− MLECs. Expression was normalized to GAPDH.…

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  Figure 7.. Plasma miR-217 is a biomarker of aging associated with cardiovascular risk in humans. A, Plasma miR-217 is a biomarker of human aging. The proportion of individuals with detectable plasma miR-217 was determined by qPCR in samples from young healthy control individuals (<50 y old; n=42) and nonagenarian individuals (n=41). B, miR-217 is a biomarker for cardiovascular aging. The proportion of individuals with detectable miR-217 in plasma stratified by lifestyle habits in young control and nonagenarian cohorts or only in the young cohort. C, Plasma miR-217 is associated with increased body mass index (BMI). BMI values are shown for individuals with detectable or no detectable plasma miR-217 in cohorts of sex-matched young healthy controls (<50 y old) and nonagenarian women. The gray line indicates overweight (BMI, ≥25). Unpaired t test P=0.05. D, Serum miR-217 is a biomarker of atherosclerosis in humans. The chart shows the proportions of healthy controls (n=34) and individuals with coronary atherosclerosis (n=32) with detectable miR-217 in plasma determined by quantitative real-time polymerase chain reaction. qPCR indicates quantitative polymerase chain reaction. *P<0.05 by Fisher…