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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Hypertension. 2021 Jun 28;78(2):282–293. doi: 10.1161/HYPERTENSIONAHA.120.15070

Targeting MicroRNA-192–5p, a Downstream Effector of NOXs, Reverses Endothelial Dihydrofolate Reductase Deficiency to Attenuate Abdominal Aortic Aneurysm Formation

Kai Huang 1,#, Taro Narumi 1,#, Yixuan Zhang 1, Qiang Li 1, Priya Murugesan 1, Yusi Wu 1, Norika Mengchia Liu 1, Hua Cai 1
PMCID: PMC8266749  NIHMSID: NIHMS1709112  PMID: 34176283

Abstract

We have shown that endothelial specific dihydrofolate reductase (DHFR) deficiency underlies eNOS uncoupling and formation of abdominal aortic aneurysm (AAA). Here we examined a novel role of miR-192–5p in mediating NOX-dependent DHFR deficiency and AAA formation. miR-192–5p is predicted to target DHFR. Intriguingly, hsa-miR-192–5p expression was substantially upregulated in human AAA patients. In human aortic endothelial cells (HAECs) exposed to hydrogen peroxide (H2O2), hsa-miR-192–5p expression was significantly upregulated. This was accompanied by a marked downregulation in DHFR mRNA and protein expression, which was restored by hsa-miR-192–5p specific inhibitor. Of note, miR-192–5p expression was markedly upregulated in Ang II infused hph-1 mice, which was attenuated in hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 double mutant mice where AAA incidence was also abrogated, indicating a downstream effector role of miR-192–5p following NOX activation. In vivo treatment with mmu-miR-192–5p inhibitor attenuated expansion of abdominal aortas in Ang II infused hph-1 mice as defined by echocardiography and post-mortem inspection. It also reversed features of vascular remodeling including matrix degradation, adventitial hypertrophy and formation of intra-lumen thrombi. These animals had restored DHFR mRNA and protein expression, attenuated superoxide production, recoupled eNOS, and preserved NO bioavailability. In conclusion, our data for the first time demonstrate a critical role of miR-192–5p in mediating NOX-dependent DHFR deficiency and AAA formation, inhibition of which is robustly effective in attenuating development of AAA. Since the mouse and human miR-192–5p sequences are identical, the miR-192–5p inhibitors may be readily translatable into novel therapeutics for the treatment of AAA.

Keywords: Abdominal aortic aneurysm (AAA), Angiotensin II (Ang II), dihydrofolate reductase (DHFR), NADPH oxidase (NOX), microRNA-192–5p (miR-192–5p), endothelial nitric oxide synthase (eNOS), oxidative stress, reactive oxygen species (ROS)

Summary

These new findings indicate that specifically targeting miR-192–5p may serve as a novel therapeutic option for the treatment and prevention of AAA.

1. INTRODUCTION

Abdominal aortic aneurysm (AAA), characterized by a localized dilatation over 30 mm in diameter, is a severe and lethal vascular disease1. Patients often do not have symptoms till late stage, and die of ruptured aneurysm suddenly, which accounts for more than 9,750 deaths per year in the United States and 200,000 deaths worldwide2. Open repair and endovascular aneurysm repair (EVAR) are the only therapeutic options for aneurysms over 5.5 cm in size3. There have been no oral medicines available to treat aneurysms smaller than 5.5 cm, resulting in silent growth of the aneurysms and lethal ruptures. While the detailed molecular mechanisms underlying AAA remain to be fully understood, a causal role of oxidative stress has been established for the formation of AAA and other types of aortic aneurysms4–13. Inflammatory and vascular cells are activated by oxidative stress to produce MMPs, resulting in matrix degradation, weakening of medial layer and formation of AAA.

We have previously shown that an endothelial cell specific dihydrofolate reductase (DHFR) deficiency underlies Angiotensin II (Ang II) induced eNOS uncoupling and eNOS uncoupling-dependent formation of AAA in Ang II infused hyperphenylalaninemia (hph)-1 mice and apoE null mice7–9, 14. In hph-1 and apoE null mice, Ang II infusion augments eNOS uncoupling via downregulation of DHFR7–9, 14. Folic acid prevents progressive uncoupling of eNOS and vascular remodeling via restoration of DHFR function, resulting in completely normalized blood pressure in WT mice and abrogated AAA formation in Ang II-infused hph-1 mice and apoE null mice7, 8. Utilizing double knockout strategies, we have further shown that DHFR deficiency lies downstream of NOX isoforms 1, 2 or 4 in Ang II-infused hph-1 mice, in line with our previous findings that hydrogen peroxide (H2O2) produced by NOX induces DHFR deficiency9, 15. Mice knockout of DHFR displayed phenotypes of more severe vascular remodeling, and exaggerated AAA and hypertension via mitochondrial dysfunction14.

microRNAs (miRs) are small, endogenous, single-stranded non-coding RNA molecules of 18–22 nucleotides which constitute a novel class of gene regulators16, 17. They bind to the 3′-untranslated regions (3′-UTRs) of specific messenger RNAs to induce their degradation or translational repression via an imperfect complement in animal cells or perfect complement in plant cells16, 17. miR-192–5p was reported to decrease DHFR protein expression via translational arrest18. Yang et al. reported that levels of miR-192–5p are reduced in medulloblastoma cells, and that DHFR is a target of miR-192–5p as evidenced by dual luciferase miR target reporter assay indicating reduced DHFR promoter activity with co-transfection of miR-192–5p mimics19. Overexpression of miR-192 by miR-192 mimics suppressed cellular viability and proliferation, and increased cell cycle arrest via downregulation of DHFR19. However, whether or not miR-192 plays a role in AAA formation via direct targeting of DHFR in endothelial cells has never been examined.

In the present study, we investigated a potential intermediate role of miR-192–5p in NOX-dependent downregulation of DHFR, and subsequent formation of AAA. The expression level of miR-192–5p was substantially upregulated in the aortic aneurysmal tissues of human AAA patients (n=15 for both AAA patients and controls), H2O2 treated human aortic endothelial cells (HAECs,) and Ang II treated hph-1 mice, while it was decreased in double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 mice. In vivo treatment with miR-192–5p specific inhibitors markedly restored DHFR mRNA and protein levels, decreased superoxide production, recoupled eNOS, restored NO bioavailability, and attenuated AAA formation. These results indicate that targeting miR-192–5p may serve as a novel therapeutic approach for the treatment and/or prevention of AAA.

2. MATERIALS AND METHODS

Data, analytic methods, and study materials will be made available to other researchers on publication of this research article. These will not be stored online or publicly but can be shared electronically or physically on request (see Materials in the online-only Data Supplement). Besides sections below, other methods paragraphs have been included in the Online Supplement 7, 9, 10, 12, 14, 20–22.

2.1. Human AAA samples

The aortic aneurysmal tissue samples of human AAA were obtained from the NIH NDRI (National Disease Research Interchange) program with approved IRB (Institutional Review Board) protocol, and the control subjects were those of donors died of sudden causes, but without aneurysms (age: 73.1±11.4 years old for control vs. 72.5±10.8 years old for AAA; 11 males and 4 females for control vs. 10 males and 5 females for AAA).

2.2. Cell culture and miRNA inhibitors transfection

Human aortic endothelial cells (HAECs) of passages 3 to 7 donated from 2 males (aged 49 and 50; Lonza; Walkersville, MD) were cultured in EGM2 media supplemented with 10% (v/v) fetal bovine serum (FBS) and supplements (Hydrocortisone, hFGF-B, VEGF, R3-IGF-1, Ascorbic Acid, hEGF, GA-1000 and Heparin, all reagents from Lonza, USA). Cells were grown in a humidified atmosphere at 5% CO2 and 37°C. miRNA inhibitors and negative controls (50 µmol/L; 100 pmol/well in six-well plate, Life Technologies Corporation, Grand Island, NY 14072, USA) transfection into HAECs was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturers’ instructions. Transfection was performed 48 h prior to being stimulated with 100 μmol/L H2O2 for 24 h in HAECs. Then cells were harvested for subsequent analyses of has-miR-192 expression, and DHFR mRNA and protein expression.

2.3. RNA extraction, microRNA-specific cDNA synthesis, and qRT-PCR of microRNA

Total RNAs were extracted from HAECs using TRIzol® (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from RNA samples using the Mir-X miRNA First-Strand Synthesis kit (Clontech Laboratories, Inc., A Takara Bio Company, Mountain View, CA, USA) according to the manufacturer’s instructions. Primers were designed on the basis of miRBase sequence (hsa-miR-192–5p, MIMAT0000222, miRBase). Variability in the initial quantities of cDNA was normalized relative to the abundance of U6 after amplification (supplied by Clontech Laboratories, Inc.), and the data were expressed as fold changes. qRT-PCR of microRNAs were conducted with the SYBR qRT-PCR kit (Clontech Laboratories, Inc., A Takara Bio Company, Mountain View, CA, USA) according to the manufacturer’s instructions.

2.4. In vivo treatment of hph-1 mice with mmu-miR-192–5p inhibitors

The locked nucleic acid (LNA)-mmu-miR-192–5p inhibitors were synthesized by Exiqon (now a QIAGEN company, Germantown, MD, USA), and used to inject (30 mg/kg each time) into hph-1 mice subcutaneously on the first day and the third day after implantation of Ang II pumps. The LNA-negative-control was injected into the animals as a control group. mmu-miR-192–5p mirVana® miRNA inhibitor and negative control for in vivo experimentation were synthesized by ThermoFisher (Grand Island, NY, USA). mmu-miR-192–5p mirVana® miRNA inhibitor or negative control was injected intravenously into hph-1 mice via tail vein on the first day and the third day (2.5 mg/kg each time) after implantation of Ang II pumps.

2.5. Electron spin resonance (ESR) Determination of Superoxide Levels

As previously described7–10, 12, 14, 15, 20, 21, 23, freshly isolated aortas were homogenized on ice in lysis buffer supplemented with protease inhibitor cocktail (1:100), and centrifuged at 12,000 g for 15 min. Protein content of the supernatant was determined using a protein assay kit (Bio-Rad, Irvine, CA, USA). Five μg of proteins were mixed with ice-cold and nitrogen bubbled KHB containing diethyldithiocarbamic acid (5 μmoL/L), deferoxamine (25 μmoL/L), and the freshly prepared superoxide specific spin trap methoxycarbonyl- 2,2,5,5-tetramethylpyrrolidine (CMH, 500 μmoL/L, Axxora, San Diego, CA, USA). The mixture was then loaded into a glass capillary (Kimble, Dover, OH, USA), and assayed using the electron spin resonance (ESR) spectrometer (eScan, Bruker, Billerica, MA, USA) for superoxide production. A second measurement was taken with the addition of PEG-SOD (100 U/mL). To assess eNOS uncoupling activity, a third measurement was made with the addition of L-NAME (100 μmoL/L). The ESR settings used were: center field, 3480; sweep width, 9 G; microwave frequency, 9.78 GHz; microwave power, 21.02 mW; modulation amplitude, 2.47 G; 512 points of resolution; receiver gain, 1000.

2.6. Electron spin resonance (ESR) determination of nitric oxide (NO) bioavailability

Aortic NO bioavailability was determined by ESR as we previously described7–10, 12, 14, 15, 20, 21, 23–25. In brief, freshly isolated aortas were cut into 2 mm rings, and then incubated in freshly prepared NO specific spin trap Fe2+(DETC)2 (0.5 mmol/L) colloid in nitrogen bubbled, modified Krebs/ HEPES buffer at 37°C for 60 min, in the presence of calcium ionophore A23187 (10 μmol/L). The aortic rings were snap frozen in liquid nitrogen and loaded into a finger Dewar for measurement with ESR spectrophotometer (eScan, Bruker, Billerica, MA, USA). The instrument settings used were as the followings: Center field, 3440; Sweep width, 100 G; microwave frequency, 9.796 GHz; microwave power 13.26 mW; modulation amplitude, 9.82 G; 512 points of resolution; and receiver gain 356.

2.7. Statistical analysis

All statistical analyses were carried out with the Prism software. Comparison between two groups was performed using the student’s t-test. Comparisons between multiple groups were done using one-way ANOVA, followed by the Newman-Keuls post-hoc test. Comparisons of the incidence rates of AAA among different animal groups were performed using chi-squared test. Statistical significance was set at p < 0.05. All grouped data are presented as Mean ± SEM.

3. RESULTS

3.1. Hydrogen peroxide downregulated DHFR expression while upregulating miR-192–5p expression

In the present study we examined an intermediate role of miR-192–5p in NOX-dependent modulation of DHFR to impact on AAA formation. First, we examined expression of miR-192–5p in aortic aneurysmal tissues of human AAA. Intriguingly, the expression of hsa-miR-192–5p was substantially upregulated in the aortic aneurysmal tissues of human AAA patients comparing to donor controls (Fig. 1A). The aortic aneurysmal tissue samples of human AAA and the control non-AAA subjects were obtained from the NIH NDRI program with approved IRB protocol (age: 73.1±11.4 years old for control vs. 72.5±10.8 years old for AAA; 11 males and 4 females for control vs. 10 males and 5 females for AAA). In H2O2 (100 μM, 24 h) treated HAECs, hsa-miR-192–5p expression was also significantly upregulated (Fig. 1B). Interestingly, DHFR is a putative target of miR-192–5p by TargetScan (http://www.targetscan.org/) (Fig. 1C). Of note, miR-192–5p was shown to decrease DHFR protein abundance in human colon cancer cell lines18, and inhibit medulloblastoma cell proliferation by binding to DHFR 3-UTR19. We therefore hypothesize that miR-192–5p, with increased expression in both aortic aneurysmal tissues of human AAA patients and H2O2 treated HAECs, might mediate H2O2 downregulation of DHFR in vivo to induce AAA formation (see below).

Figure 1. miR-192–5p, which is predicted to target DHFR, is upregulated in human AAA and hydrogen peroxide treated human aortic endothelial cells (HAECs).

Figure 1.

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The RNA was isolated from aortic samples of human AAA patients and control subjects (n=15 for each group). A. miR-192–5p was upregulated in aortic samples of human AAA patients comparing to the controls. Data are presented as Mean±SEM (n=15), **p<0.01 vs. control. Human aortic endothelial cells (HAECs) were treated with hydrogen peroxide (H2O2, 100 µM, 24 h) and the RNA was isolated. B. Hsa-miR-192–5p expression was upregulated in H2O2 stimulated HAECs. Data are presented as Mean±SEM (n=4–6), *p<0.05 vs. control. TargetScan was used to explore the putative relationship between human miR192–5p and dihydrofolate reductase (DHFR). C. DHFR is predicted to be the putative target of miR192–5p by TargetScan.

3.2. Silencing of miR-192–5p with specific inhibitors restored DHFR expression in endothelial cells

To examine whether miR-192–5p downregulates DHFR in ECs, HAECs were exposed to H2O2 after treansfection of miR-192–5p inhibitors for 48 h. As shown in Fig. 2A, miR-192–5p expression levels were reduced in miR-192–5p-specific inhibitor treated HAECs. Furthermore, hsa-miR-192–5p-specific inhibitor markedly restored DHFR mRNA (Fig. 2B) and protein expression (Fig. 2C and 2D), indicating an intermediate role of miR-192–5p in H2O2 induced DHFR deficiency.

Figure 2. Hsa-miR-192–5p specific inhibitors restore DHFR mRNA and protein expression in hydrogen peroxide stimulated HAECs.

Figure 2.

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Human aortic endothelial cells (HAECs) were treated with hydrogen peroxide (H2O2, 100 µM, 24 h) in the presence or absence of negative control miR inhibitors or Has-miR-192–5p inhibitors, and the RNA and protein were isolated for RT-PCR and Western blotting analyses to detect miR-192–5p and DHFR mRNA and protein expression levels. A. Hsa-miR-192–5p specific inhibitors attenuated miR-192–5p expression in H2O2 stimulated HAECs. Data are presented as Mean±SEM (n=6), *p<0.05. B. Hsa-miR-192–5p specific inhibitors markedly restored DHFR mRNA expression in H2O2 stimulated HAECs. Data are presented as Mean±SEM (n=4–6), **p<0.01, ***p<0.001. C. Representative Western blots of endothelial DHFR protein expression with β-actin serving as an internal control. D. Hsa-miR-192–5p specific inhibitors markedly restored DHFR protein expression in H2O2 stimulated HAECs. Data are presented as Mean±SEM (n=4), *p<0.05.

3.3. miR-192–5p expression in Ang II infused hph-1 mice: NOX-dependent upregulation

Of note, miR-192–5p is highly conserved among species, especially in the seed region (red part) that is predicted to target DHFR (supplemental Fig. S1). Therefore, we explored a potential role of miR-192–5p in AAA formation in Ang II-infused hph-1 mice, via predicted downregulation of DHFR in vivo. We have previously shown that DHFR deficiency lies downstream of NOX isoforms 1, 2 or 4 activation in Ang II-infused hph-1 mice, resulting in eNOS uncoupling to induce AAA formation9. The incidence of AAA was substantially reduced, with significant difference by chi-square test, from 79.2% in AngII-infused hph-1 animals to 11.8%, 15.2%, 7.7% and 0% in hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 double mutant animals, respectively (Fig. 3A and 3B) (combined data from the present study and the previous work9). The miR-192–5p expression was significantly upregulated in Ang II infused hph-1 mice compared to WT mice, which was abrogated in hph-1-NOX1 (Fig. 3C), hph-1-NOX2 (Fig. 3D), hph-1-p47phox (Fig. 3E) and hph-1-NOX4 (Fig. 3F) double mutant mice, indicating a downstream role of miR-192–5p following NOX activation.

Figure 3. miR-192–5p serves as a downstream effector of NOXs in mediating AAA formation.

Figure 3.

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Ang II was infused into hph-1, hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 double mutant animals prior to phenotyping for AAA and isolation of aortic endothelial cells for detection of miR-192–5p expression levels. A. The percentage of AAA in each experimental groups. n=26–53, ***p<0.001. B. The actual numbers of animals in each experimental groups with and without AAA, combined with our original data reported in Siu KL et al. Redox Biol. 2017;11:118–125. The incidence of AAA was greatly reduced from 79.2% in Ang II-infused hph-1 mice to 11.8%, 15.2%, 7.7% and 0% in hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 double mutant animals respectively. C. The mmu-miR-192–5p expression levels in hph-1 and hph-1-NOX1 mice with and without Ang II infusion. Data are presented as Mean±SEM (n=5), *p<0.05, **p<0.01. D. The expression levels of mmu-miR-192–5p in hph-1 and hph-1-NOX2 mice with and without Ang II infusion. Data are presented as Mean±SEM (n=6–7), *p<0.05. E. The expression levels of mmu-miR-192–5p in hph-1 and hph-1-p47phox mice with and without Ang II infusion. Data are presented as Mean±SEM (n=4), ***p<0.001. F. The expression levels of mmu-miR-192–5p in hph-1 and hph-1-NOX4 mice with and without Ang II infusion. Data are presented as Mean±SEM (n=6), **p<0.01, ***p<0.001.

3.4. miR-192–5p inhibitors restored DHFR expression in Ang II infused hph-1 mice

Of note, the mmu-miR-192–5p specific inhibitors were used to examine whether inhibition of miR-192–5p restores DHFR expression in Ang II infused hph-1 mice. As demonstrated in Fig. 4A, qRT-PCR analysis of miR expression indicated that mmu-miR-192–5p specific inhibitors decreased mmu-miR-192–5p expression in ECs isolated from aortas of Ang II infused hph-1 mice. Ang II infused hph-1 mice had significant decreased DHFR mRNA (Fig. 4B) and protein expression (Fig. 4C and 4D) compared to hph-1 mice, while mmu-miR-192–5p inhibitors substantially restored DHFR mRNA (Fig. 4B) and protein expression (Fig. 4C and 4D) comparing to Ang II infused hph-1 mice in ECs isolated from aortas.

Figure 4. Mmu-miR-192–5p specific inhibitors restore DHFR mRNA and protein expressions in Ang II-infused hph-1 mice.

Figure 4.

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Mmu-miR-192–5p-specific inhibitors and negative controls were injected into Ang II infused hph-1 mice, and aortic endothelial cells were harvested to detect miR-192–5p expression, and DHFR mRNA and protein expression levels. A. Mmu-miR-192–5p-specific inhibitors attenuated mmu-miR-192–5p expression in Ang II infused hph-1 mice. Data are presented as Mean±SEM (n=6), **p<0.01, ***p<0.001. B. Mmu-miR-192–5p specific inhibitors markedly restored DHFR mRNA in Ang II infused hph-1 mice. Data are presented as Mean±SEM (n=4–6), **p<0.01, ***p<0.001. C. Representative Western blots of endothelial DHFR protein expression with β-actin serving as an internal control. D. Mmu-miR-192–5p specific inhibitors markedly restored DHFR protein expression in Ang II infused hph-1 mice. Data are presented as Mean±SEM (n=4), *p<0.05.

3.5. miR-192–5p inhibitors diminished endothelial superoxide production, recoupled eNOS and restored NO bioavailability in Ang II infused hph-1 mice

We have previously shown that endothelial DHFR deficiency leads to a reduction in H4B bioavailability and consequent endothelial nitric oxide synthase (eNOS) uncoupling to result in development of AAA7–10, 14. Aortic production of total ROS detected by DHE staining was markedly increased in Ang II infused hph-1 mice comparing to untreated hph-1 mice, which was significantly attenuated in Ang II infused hph-1 mice with mmu-miR-192–5p inhibitors (Fig. 5A and 5B). In addition, aortas were harvested and subjected to electron spin resonance (ESR) determination of superoxide production in the presence or absence of L-NAME, an inhibitor of NOS. If eNOS is functional and coupled, its inhibition by L-NAME to remove the buffering effect of NO will increase the measured superoxide. However, if eNOS is dysfunctional and uncoupled, it produces superoxide. Therefore, inhibition with L-NAME will reduce measured superoxide. There is an increased superoxide production in Ang II infused hph-1 mice, and this increase was inhibited by L-NAME, indicating that uncoupled eNOS is the enzymatic source of the superoxide production (Fig. 5C), as we previously shown7–10, 14. L-NAME-sensitive superoxide production, reflective of eNOS uncoupling activity, was completely attenuated by miR-192–5p inhibitors (Fig. 5C). Notably, NO bioavailability was decreased in Ang II infused hph-1 mice, which was also substantially restored by miR-192–5p inhibitors (Fig. 5D). These results show that miR-192–5p inhibitors could improve the coupling state of eNOS, reduce ROS production and restore NO bioavailability in Ang II infused hph-1 mice via restoration of DHFR expression. Our previous work 7–10, 14 and these data again confirm that in addition to vascular smooth muscle (VSMC) generation of superoxide in response to Ang II, uncoupled eNOS can serve as a major contributor of endothelial specific production of superoxide production to result in AAA formation (also Fig. 6 below).

Figure 5. In vivo treatment with mmu-miR-192–5p specific inhibitors attenuates ROS production, recouples eNOS and restores NO bioavailability.

Figure 5.

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Mmu-miR-192–5p-specific inhibitors and negative controls were injected into Ang II infused hph-1 mice, and the aortas were freshly harvested for dihydroethidium (DHE) imaging analysis of superoxide production, electron spin resonance (ESR) analyses of superoxide production and eNOS uncoupling activity, and ESR determination of NO bioavailability. A. Aortic production of superoxide detected by DHE imaging was markedly increased in Ang II infused hph-1 mice, which was substantially attenuated by in vivo treatment with mmu-miR-192–5p specific inhibitors. B. Quantitative analysis of fluorescent intensity of DHE images, indicating same notion as in A. Data are presented as Mean±SEM (n=4–5), **p<0.01, ***p<0.001. C. Total superoxide production determined by ESR in the presence or absence of L-NAME (NOS inhibitor). There is a very modest eNOS uncoupling (L-NAME-inhibitable superoxide production) activity at baseline in hph-1 mice as we previously published. The marked increase in eNOS uncoupling activity in Ang II-infused hph-1 mice was completely attenuated by in vivo treatment with mmu-miR-192–5p specific inhibitors. Data are presented as Mean±SEM (n=6–7), *p<0.05 vs. L-NAME(−) for all corresponding groups, #p<0.05 vs. Sham group without Ang II infusion, & p<0.05 vs. Ang II and Ang II + negative control groups without L-NAME. D. NO bioavailability determined by ESR was markedly decreased in Ang II infused hph-1 mice, which was significantly restored by in vivo treatment with mmu-miR-192–5p specific inhibitors. Data are presented as Mean±SEM (n=7–9), *p<0.05, **p<0.01.

Figure 6. mmu-miR-192–5p specific inhibitors attenuate AAA development in Ang II infused hph-1 mice.

Figure 6.

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Mmu-miR-192–5p-specific inhibitors and negative controls were injected into Ang II infused hph-1 mice, prior to phenotyping of the mice for AAA formation. A. The percentage of AAA in Ang II infused hph1 mice treated with negative controls and mmu-miR-192–5p specific inhibitors. The incidence of AAA was greatly reduced from 80.0% in hph-1 animals treated with negative controls, to 25.0% in hph-1 mice treated with mmu-miR-192–5p specific inhibitors. n=10–20, ***p<0.001. B. Time-dependent expansion of abdominal aortas defined by echocardiography was attenuated by in vivo treatment with miR-192–5p specific inhibitors in Ang II infused hph-1 mice. n=7, **p<0.01, ***p < 0.001 vs. sham; #p<0.05 vs. Ang II; &p<0.05 vs. Ang II + negative control. C. Post-mortem inspection indicated that AAA formation was attenuated by in vivo treatment of mmu-miR-192–5p specific inhibitors in Ang II infused hph-1 mice. D. Ang II infusion into hph-1 mice induced a marked adventitial hypertrophy and intra-wall thrombosis, which was attenuated by in vivo treatment with mmu-miR-192–5p specific inhibitors. E. Ang II infusion into hph-1 mice induced enlargement of external dimeters of the abdominal aortas, which was attenuated by in vivo treatment with mmu-miR-192–5p specific inhibitors. Data are presented as Mean±SEM (n=10–11), *p<0.05, ***p < 0.001. F. VVG staining indicated significant degradation and flattening of elastic fibers in the aortic medial layers of Ang II infused hph-1 mice (red arrows), which was restored by in vivo treatment with mmu-miR-192–5p specific inhibitors.

3.6. miR-192–5p inhibitors attenuated AAA formation in Ang II infused hph-1 mice

Since miR-192–5p expression was increased in Ang II infused hph-1 mice, we explored a potential intermediate role of miR-192–5p in the development of AAA via downregulation of DHFR. The incidence of AAA was greatly reduced from 80.0% in negative control treated hph-1 animals to 25.0% in miR-192–5p specific inhibitors treated hph-1 animals after Ang II infusion (Fig. 6A). At days 0, 7, and 14, abdominal ultrasound was performed to assess abdominal aorta (AA) dimensions. The sizes of AA measured by echocardiography were significantly smaller in hph-1 mice treated with mmu-miR-192–5p inhibitors, compared to that of control group (Fig. 6B). Post-mortem inspection indicated that AAA formation was prevented in Ang II infused hph-1 mice with in vivo treatment of mmu-miR-192–5p inhibitors (Fig. 6C). Representative images of H&E staining were shown in Fig. 6D, which confirmed that mmu-miR-192–5p inhibitors attenuated AAA formation in Ang II infused hph-1 mice. Besides, the external diameters of the AAA were increased in negative control treated hph-1 animals, which were attenuated in miR-192–5p specific inhibitors treated hph-1 mice after Ang II infusion (Fig. 6E). Of note, Ang II infusion induced marked adventitial hypertrophy as well as intra-wall thrombosis, which were markedly attenuated by mmu-miR-192–5p inhibitors. VVG staining indicated significant degradation and flattening of elastic fibers in Ang II infused hph-1 mice, which was substantially abrogated by in vivo treatment with miR-192–5p inhibitors (Fig. 6F). Notably, the sequences of miR-192–5p are the same between human and mouse (Fig. S1). Therefore, these results indicate that miR-192–5p inhibitors maybe be readily used as potential therapeutics for human AAA.

4. DISCUSSION

The most significant findings in the present study are the first demonstration that miR-192–5p plays a critical role in mediating NOX-dependent DHFR deficiency in AAA formation, and that in vivo silencing of miR-192–5p expression with specific inhibitors is markedly effective in preventing AAA formation via preservation of endothelial DHFR expression, coupling activity of eNOS and NO bioavailability in ECs. Our data indicate that H2O2 generated from NOXs activates miR-192–5p expression to reduce DHFR protein abundance, resulting in eNOS uncoupling-dependent AAA formation. Inhibition of miR-192–5p in vivo with specific inhibitors restored endothelial DHFR expression and coupling activity of eNOS to result in reduced oxidative stress, restored NO bioavailability and prevention of matrix degradation and adventitial hypertrophy, hallmarks of AAA formation. Therefore, miR-192–5p may serve as a novel target for the treatment of AAA.

AAA is a progressive vascular disease, and several miRNAs have been implicated in the pathogenesis of AAA26, 27. miR-33a-5p expression in central zone of human AAA is higher than marginal zone, and that miR-33 deletion attenuated AAA formation in mice via downregulation of MMP9 in macrophages and monocyte chemotactic protein-1 in VSMCs28. miR-155 expression was found significantly increased in AAA biopsies, while circulating miR-155 levels were also elevated in AAA patients compared with controls, with a 2.67-fold up-regulation at borderline significance29. Two immunologically important miR-155 target genes, CTLA4 (cytotoxic T-lymphocyte-associated protein) and SMAD2 (homologies to the Caenorhabditis elegans SMA and MAD family of genes in Drosophila) were found significantly down-regulated within AAA bodies compared with AAA necks, which play an important role in promoting chronic inflammation by enhancing T-cell development and decreasing expression of TGF-β-dependent genes in the nucleus29. However, the detailed molecular mechanisms of miRNAs in human AAA need to be explored further, not to mention that the specific roles and regulations of endothelial miRNAs. Here, our data for the first time demonstrated that endothelial miR-192–5p was upregulated in aortic aneurysmal tissues of human AAA, and it played a critical role in mediating AAA formation in Ang II infused hph-1 mice via downregulation of DHFR and consequent uncoupling of eNOS. Inhibition of miR-192–5p in vitro and in vivo restored DHFR expression and eNOS coupling activity to result in abrogation of AAA formation.

DHFR deficiency uncouples eNOS to induce hypertension and AAA formation7–10, 13–15, 20, 23. Relatively modest DHFR deficiency results in a two-fold increase in eNOS uncoupling activity and development of hypertension in Ang II infused WT mice, while more severe DHFR deficiency in Ang II infused hph-1 mice induces a three-fold eNOS uncoupling activity to lead to AAA formation7. We have previously established that augmentation of endothelial DHFR expression and activity is robustly effective in protecting against development of AAA7–10, 13, 14. Here, we have shown for the first time that miR-192–5p was able to induce DHFR deficiency in human endothelial cells in vitro and Ang II infused hph-1 mice in vivo. Besides, miR-192–5p inhibitors restored DHFR mRNA and protein expression in H2O2 stimulated endothelial cells and in Ang II-infused hph-1 mice to attenuate eNOS uncoupling activity. Intriguingly, miR-192–5p inhibitors reduced the incidence rate of AAA from 80% to 25%, and substantially attenuated AAA formation at molecular and histological levels in Ang II infused hph-1 mice. The miR-192–5p inhibitors abrogated vascular remodeling including medial elastin degradation and flattening, as well as adventitial hypertrophy, features shown by our previous studies to characterize AAA formation in Ang II infused hph-1 mice. Whether regulation of miR-192–5p in endothelial cells plays a role in adventitial hypertrophy in other vascular diseases remain to be further investigated. Overall, our data establish an important role of miR-192–5p in the formation of AAA in the robust model of Ang II infused hph-1 mice. Notably, the sequences of miR-192–5p are the same between human and mouse. Therefore, miR-192–5p inhibitors maybe are readily used as potential powerful therapeutics for human AAA.

NOX isoform 1, 2 or 4 lies upstream of DHFR deficiency to induce AAA formation9. Activation of NOX by Ang II produces ROS to contribute to cardiovascular pathogenesis13, 30–34. NOX produces ROS in response to Ang II in endothelial cells and vascular smooth muscle cells (VSMCs)33, 34. Endothelial NOX-derived H2O2 down-regulates DHFR expression in response to Ang II15. Double mutant mice of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4 had preserved DHFR expression and activity in endothelial cells in response to Ang II infusion9. Of note, miR-192–5p expression was significantly increased in Ang II infused hph-1 mice, which was significantly abrogated in Ang II infused hph-1-NOX1, hph-1-NOX2, hph-1-p47phox and hph-1-NOX4 mice, indicating downstream role of miR-192–5p in mediating NOX-dependent DHFR deficiency. Mmu-miR-192–5p specific inhibitors restored DHFR mRNA and protein expression in Ang II-infused hph-1 mice. Of note, animal and preliminary human data have shown that miRNA mimics and inhibitors have the great potential to develop into a whole new class of therapeutics for the treatment of cardiovascular diseases35–38. miRNAs are small RNA molecules with known sequence that is often remarkably conserved between species, such as miR-192–5p in this research. These characteristics make miRNAs excellent drug targets that can be manipulated with mostly on-target effects, which have promoted miRNA-modulating compounds to enter preclinical efficacy and safety studies as well as in clinical trials39, 40. anti-miRs can be generated based on antisense technologies, and can effectively bind to their cognate miRNA targets with excellent affinity and specificity39–41; the mmu-miR-192–5p inhibitors used in this study have indeed proved to be highly efficient and effective in vivo in attenuating AAA formation.

In conclusion, our work represents the first evidence that miR-192–5p, downstream of activation of NOX isoforms in response to Ang II, mediates H2O2 induced endothelial DHFR deficiency, eNOS uncoupling and consequent AAA formation. Whereas, specific inhibition of miR-192–5p in Ang II infused hph-1 mice is robustly effective in attenuating AAA formation via preservation of endothelial DHFR expression and eNOS coupling activity, and abrogation of sustained oxidative stress, matrix degradation and vascular remodeling. Since the human and the mouse miR-192–5p sequences are identical, these data indicate that miR-192–5p inhibitors may be readily used as novel therapeutic options for AAA.

Perspectives

As schematically illustrated in the Graphic Abstract, Ang II induces a rapid and transient activation of endothelial NOX, resulting in oxidative stress and H2O2-dependent upregulation of miR-192–5p, which in turn leads to decreased DHFR mRNA and protein expression, uncoupling of eNOS, and development of AAA. Inhibition of miR-192–5p with antagomiR-192–5p, however, attenuates AAA formation in Ang II–infused hph-1 mice, via upregulation of DHFR protein expression to restore NO bioavailability and coupling activity of eNOS. Indeed, miR-192–5p expression is upregulated in aneurysmal aortic samples of human AAA patients, and the sequences of miR-192–5p are identical between human and mouse. These findings strongly support the robustness and translational value of our molecular mechanistic findings. In conclusion, our work in the present study indicates that specifically targeting miR-192–5p may be used as a novel therapeutic option for the treatment and prevention of AAA.

Supplementary Material

Supplemental Publication Material

Novelty and Significance.

What Is New?

  • First identification of an intermediate role of miR-192–5p in dihydrofolate reductase (DHFR) deficiency and abdominal aortic aneurysm (AAA) formation downstream of NADPH oxidase (NOX) activation.

  • First identification that targeting miR-192–5p to restore DHFR and endothelial nitric oxide synthase (eNOS) coupling activity is highly effective in attenuating AAA formation.

What Is Relevant?

  • Our data reveal a novel mechanism underlying oxidative stress-induced AAA formation.

  • Our data establish a novel therapeutic approach for the treatment and prevention of AAA.

Acknowledgments

Sources of Funding

This study was supported by NIH National Heart, Lung, and Blood Institute (NHLBI) grants HL077440 (H.C.), HL088975 (H.C.), HL142951 (H.C.) and HL154754 (H.C.).

Footnotes

Disclosures

None.

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