Endothelial MicroRNA-214 Confers Angiotensin II Hypertension by Targeting eNOS in Mice

Figures

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  Fig. 1.. Antagonism of miR-214 after Ang II infusion attenuated Ang II hypertension in wild-type mice. a Relative miR-214 levels detected in aortas of Ang II-treated mice (n = 5–9). b qRT-PCR analysis of miR-214 expression in the MAECs treated with Ang II at various doses (0.1–5 μM for 24 h; n = 3). c SBP in Ang II-induced mice with anti-control or anti-miR-214 treatment (two-way ANOVA; n = 7). d Western blotting analysis of eNOS/p-eNOS proteins in aortas. e Western blot quantification of eNOS/p-eNOS (n = 5–6). Hypertension was induced in wild-type mice after injection of Ang II (1.4 mg/kg/day) by using osmotic mini-pump. After 3 days of Ang II infusion, mice were given intraperitoneal injection of miR-214 inhibitors (anti-miR-214, 10 mg/kg) every 5 days. Aorta tissues were collected after Ang II infusion. SBP was detected by the tail-cuff method. Data are presented as means ± SEM. For all experiments, when p < 0.05, there was a statistical difference between the two groups. SBP, systolic blood pressure.

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  Fig. 2.. Generation and characterization of EC-specific miR-214-deficient mice. a Strategy for the conditional targeting of miR-214. The targeted allele and loxP loci were shown. Genotypes of heterozygous (heter) and homozygous (homo) knockout mice were detected with PCR. b, c qRT-PCR detected the specific deletion of miR-214 in aortic endothelium and smooth muscle part (n = 4). d Body weight of endothelial miR-214 cKO mice and their littermate WT controls from 2 weeks to 2 months old (n = 9). e Aorta morphology was analyzed using hematoxylin-eosin (HE) staining in endothelial miR-214 cKO mice and littermate WT controls at 3 months old (n = 4). f SBP of endothelial miR-214 cKO mice and littermate controls was detected by the tail-cuff method (n = 6). No difference of the baseline systolic blood pressure was found between genotypes. Data were expressed as means ± SEM. p < 0.05 was considered statistically significant.

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  Fig. 3.. Deletion of miR-214 in endothelium attenuated Ang II-induced hypertension. a SBP was detected by the tail-cuff method. After 7 days’ habitual practice, the baseline SBP in endothelial miR-214 knockout (n = 11) and littermate control (n = 12) mice were measured. Then, the mice were transfused with Ang II using osmotic mini-pump, and the systolic blood pressure was measured (assessed by two-way ANOVA). b–d Daily MAP, SBP, and DBP in endothelial miR-214 KO (n = 7) and littermate WT control (n = 6) mice in response to 7-day Ang II infusion were detected via a telemetry system (assessed by two-way ANOVA). e Western blotting detection of eNOS and p-eNOS proteins in mice aortas after Ang II infusion (n = 8). f Quantification of the Western blots of eNOS/p-eNOS. g Urine NO levels detected by the ELISA Kit (n = 5–6). h Urinary albumin excretion in EC-specific miR-214-deficient mice after infusion of Ang II (assessed by one-way ANOVA; n = 6–12). All experiments were performed with the injection of Ang II by osmotic micropump at a dose of 1.4 mg/kg/day. Values were shown as means ± SEM. Difference between groups was considered statistically significant when p < 0.05. DBP, diastolic blood…

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  Fig. 4.. miR-214 regulated vascular eNOS in vivo. a miR-214 expression in aortas of wild-type mice after injection of miR-214 agomir. b Effect of miR-214 agomir on the mRNA expression of eNOS in mouse aortas (n = 6). c Effect of miR-214 agomir on the expressions of eNOS/p-eNOS. d Quantitative analysis of eNOS/p-eNOS western blots (n = 4–5). e SBP was detected by the tail-cuff method after the mice were given an intraperitoneal injection of miR-214 agonists (n = 5). f The effect of miR-214 antagomir on the expression of miR-214 in mouse aortas analyzed by qRT-PCR. g miR-214 antagomir effect on the mRNA level of eNOS in mouse aortas (n = 6). h Western blotting analysis of eNOS/p-eNOS protein expressions after miR-214 antagomir treatment. i Quantification of the Western blots of eNOS/p-eNOS (n = 4–5). j SBP was detected by the tail-cuff method after the mice were given an intraperitoneal injection of miR-214 inhibitors (n = 5). C57BL/6 mice were injected with miR-214 inhibitors and agonists, respectively (10 mg/kg, i.p.), and then aorta tissues were collected after 4 days of treatments. Values were presented as means ± SEM. Difference between groups was considered statistically significant…

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  Fig. 5.. miR-214 directly regulated eNOS in MAECs. a Compared with cells transfected with negative controls, the expression of miR-214 was significantly upregulated in MAECs transfected with miR-214 mimics (n = 3). b Relative mRNA expression of eNOS was downregulated in miR-214 mimics-transfected MAECs (n = 3). c The protein levels of eNOS/p-eNOS were downregulated in miR-214 mimics-transfected MAECs. d Western blot quantitative analysis of eNOS and p-eNOS in c. GAPDH was used as a control (n = 3–4). e Relative expression of miR-214 in miR-214 inhibitor- and anti-control-transfected MAECs (n = 3). f eNOS mRNA expression was upregulated in miR-214 inhibitor-transfected MAECs (n = 3). g The protein levels of eNOS/p-eNOS in miR-214 inhibitor- and anti-control-transfected MAECs. h Western blot quantitative analysis of eNOS and p-eNOS in g. GAPDH was used as loading control (n = 4). i The miR-214 sequence was compared and its binding site in eNOS mRNA 3′UTR was predicted. j The assay of luciferase in the mouse ECs cotransfected with eNOS luciferase reporter (WT) or mutant eNOS luciferase reporter (MUT) and miR-214 mimics (40 nm) for 24 h (n = 12 for both the native and mutated UTRs). Values…

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  Fig. 6.. VSMC-specific knockout of miR-214 did not affect hypertension caused by Ang II. a Strategy for the generation of VSMCs-specific miR-214 knockout mice. Genotypes of heterozygous (heter) and homozygous (homo) knockout mice were detected with PCR. b Knockout efficiency of miR-214 in VSMCs determined by qRT-PCR analysis (n = 6). c The expression of miR-214 in endothelium determined by qRT-PCR analysis (n = 6). d Body weight of miR-214 cKO mice and their littermate WT controls from 2 weeks to 2 months old (n = 8). e Aorta morphology of smooth muscle cell-specific miR-214 knockout mice and their littermate controls detected by HE staining (n = 4). f Systolic blood pressure of VSMCs-specific miR-214 knockout mice and their littermate WT controls were tested by the tail-cuff method (two-way ANOVA; n = 6). Ang II was infused by osmotic micropump at a dose of 1.4 mg/kg/day. Values were shown as means ± SEM. For all experiments, difference between groups was considered statistically significant when p < 0.05.

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  Fig. 7.. Renal proximal tubule-specific knockout of miR-214 did not affect hypertension caused by Ang II. a SBP of renal proximal tubular cell-specific miR-214 knockout mice and the control ones were measured by the tail-cuff method (two-way ANOVA; n = 6). Terminal urine volume (b), urine sodium (c), potassium (d), and chloride (e) in WT and renal proximal tubular cell-specific miR-214 cKO mice before and after Ang II treatment (two-way ANOVA; n = 5–6). f Body weight of miR-214 cKO mice and their littermate WT controls of 2 months old (two-way ANOVA; n = 5–6). Ang II was infused by osmotic micropump at a dose of 1.4 mg/kg/day. Values were shown as means ± SEM. For all experiments, difference between groups was considered statistically significant when p < 0.05.