HAECs were purchased from Cell Applications, Inc. (San Diego, CA, USA) and cultured in endothelial cell growth medium (Cell Applications, Inc.) according to the manufacturer's recommendations. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. High glucose (HG, 25 mM) was added to HAECs for 24 and 48 h in the different experiments. Mannitol (25 mM) was the osmotic control. The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD, USA), and maintained in RPMI 1640 culture medium supplemented with 10% FBS, L-glutamine, and penicillin.

Expression of mRNA in the HAECs was analyzed by real-time PCR as previously described (Wang et al., 2010). The primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vascular cell adhesion protein 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) are provided in Supplemental Data 1.

Protein expression levels in the HAECs were analyzed by western blot as previously described (Wang et al., 2010). Antibodies against IRAK-1 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at 1:1,000.

In adhesion experiments, THP-1 cells were labeled with calcein acetoxymethyl ester (Calcein-AM; Molecular Probes, Eugene, OR, USA) as previously described (Wang, H. J. et al., 2014). Briefly, THP-1 cells were stained with the dye at a concentration of 7.5 μM for 30 min immediately preceding the adhesion assay. HAECs were maintained in 12 well-plates until 90% confluence. The HAECs (10⁵ cells/well) were then treated with HG for 24 and 48 h and incubated with culture medium containing the labeled THP-1 cells (THP-1/HAECs = 7) for 10 min. Non-adherent THP-1 cells were removed by washing with PBS for 20 s. Adherent THP-1 cells were visualized and quantified in 10 randomly viewed fields by the fluorescent microscope (OLYMPUS, Japan).

The TaqMan® Array Human MicroRNA A Card V2 (Applied Biosystems, Foster, CA, USA) was used to analyze miR expression profiles. The card contains 377 preloaded human miR targets and four endogenous controls. For each sample, 500 ng of total RNA was used for reverse-transcription, using Megaplex RT primer Pool A and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNA was diluted, mixed with TaqMan Gene Expression Master Mix (Applied Biosystems), and loaded into the ports on microfluidic cards. The cards were briefly centrifuged for 1 min at 1,600 × g to distribute samples to the multiple wells, sealed to prevent well-to-well contamination, and analyzed using a 7900 HT Real-Time PCR System (Applied Biosystems).

The protocols for miR extraction and determination of different miR expression levels from HAECs were as previously described (Wang et al., 2013). The reverse transcription and PCR primer sequences are provided in Supplemental Data 1.

Selected miR mimics, inhibitors, and a negative control (NC) were transfected into HAECs as previously described (Wang, H. J. et al., 2014). After transfection, HAECs were treated with HG for 48 h, after which the expression levels of IRAK-1 mRNA, IRAK-1 protein, VCAM-1 mRNA, and ICAM-1 mRNA were determined. THP-1 adhesion assays were also performed after miR-146a-5p mimic transfection.

A partial IRAK-1 mRNA 3′-UTR containing the miR-146a-5p target site was constructed into a pGL-3-promoter vector (Promega, Madison, WI). HAECs were cotransfected with 1 μg of constructed plasmids and 100 nM of miR-146a-5p mimic and the negative control using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA). Empty vector was used as blank control. After 24 h of transfection, cells were harvested to measure luciferase activity using the Luciferase Assay System Kit (Promega, E1500), according to the manufacturer's instructions.

The HAECs were transfected with 100 nM of either ON-TARGETplus SMARTpool Human IRAK-1 small interfering RNA (siRNA; Dharmacon, Thermo Scientific, Lafayette, CO, USA) or a negative control, as previously described (Wang et al., 2016). The IRAK-1 siRNA target sequences were shown in Supplemental Data 2. Briefly, HAECs were transfected using Lipofectamine™ 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) in M-199 medium for 2 h. After transfection, the medium was changed to endothelial cell growth medium, and the HAECs were treated with HG for 48 h. After HG treatment, expression of IRAK-1 mRNA, IRAK-1 protein, VCAM-1 mRNA, and ICAM-1 mRNA were determined. THP-1 adhesion assays were also performed after IRAK-1 siRNA transfection.

The animal study was conducted with the ethical standards of the field and performed in accordance with the ethical guidelines provided by the Hungkuang University Institutional Animal Care and Use Committee (Permit Number: HK 105-02). Male db/db diabetic mice were purchased from National Laboratory Animal Center (Nangang, Taipei, Taiwan). Eleven-week db/db mice were administered (100 μL) miR-146a-5p mimic or a negative control (13 μg per week, 3 times) by tail-vein injection, using equal volume mixtures of Lipofectamine™ 2000 and miR-146a-5p mimic or negative control. The control db/db group received equal volume mixtures of vehicle (Lipofectamine™ 2000) and PBS. Three weeks later, mice were euthanized by CO₂ narcosis. The aortic tissue were carefully excised and fixed with 10% formalin solution. Paraffin sections (5 μm thickness) of aorta were prepared for immunohistochemistry (IHC) staining.

For aortic tissue sections from db/db mice, 3,3′-diaminobenzidine staining was performed using a Bond-Max autostainer (Leica Microsystems). Briefly, paraffin-embedded aortic tissue sections were placed in Tris buffered saline with Tween-20, then rehydrated through serial dilutions of alcohol, and washed with PBS (pH 7.2). Slides were then stained with primary antibodies against IRAK-1 (dilution 1:50, mouse monoclonal antibody, Santa Cruz), or ICAM-1 (dilution 1:50, mouse monoclonal antibody, Thermo Fisher), or incubated with PBS (as a negative control) on a fully automated Bond-Max system using onboard heat-induced antigen retrieval and a VBS Refine polymer detection system (Leica Microsystems).

Statistical analysis was performed using the SPSS 12.0 statistical software package for Windows (SPSS Inc., Chicago, IL, USA). All data are presented as the mean ± SEM. Independent experiments were performed to evaluate significant differences between the control and other experimental groups. Significant differences were determined using one-way analysis of variance (ANOVA) with post-hoc Tukey test or Student's t-tests, where appropriate. Significant differences were defined as p < 0.05.

We first determined the effects of HG on endothelial IRAK-1 expression. After 24 and 48 h stimulation, HG caused significant (1.12- and 1.29-fold) increases in IRAK-1 gene expression in HEACs, compared to the unstimulated control (Figure 1A). The expression of IRAK-1 protein also displayed a time-dependent increase, with 1.67- and 1.96-fold increases after 24 and 48 h of HG stimulation, respectively (Figure 1B). The osmotic control experiments showed that mannitol treatment did not modulate the expression levels of IRAK-1 (Supplemental Data 3). The adhesion of monocytes to the inflamed endothelium is a hallmark of the initiation of atherosclerotic plaques (Tuttolomondo et al., 2012). Vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) are essential molecules for this adhesive process. As shown in Figure 1C, HG stimulation for 24 and 48 h caused 1.48- and 1.88-fold increases of VCAM-1 gene expression, respectively. Similarly, HG caused 1.34- and 1.77-fold increases in ICAM-1 gene expression (Figure 1D). The increases of VCAM-1 and ICAM-1 expression levels were associated with increased adhesion of THP-1 monocytic cells to HAECs, as HG stimulation for 24 and 48 h caused 1.50- and 2.86-fold increases in THP-1 adhesion to HAECs, respectively (Figure 1E).

To determine potential binding partners of the 3′-UTR of human IRAK-1 mRNA, in silico analyses using miRanda-mirSVR (http://www.microrna.org) were performed. This online database incorporated a mirSVR scoring system to improve predictions of the effects that a miR may have on gene expression (Betel et al., 2010). A total of 13 miRs were predicted to possess homology with the 3′-UTR of the human IRAK-1 mRNA (Supplemental Data 4). The TaqMan® Array Human MicroRNA Card contained all 13 possible miRs predicted to target IRAK-1, and 24 h HG-stimulation caused the downregulation of seven endothelial miRs: miR-146a-5p, miR-339-5p, miR-874-3p, miR-125-3p, miR-431-5p, miR-192-5p, and miR-215-5p (Figure 2A). Real-time PCR analyses of 24 h HG-stimulated HAECs showed that only miR-146a-5p, miR-339-5p, miR-874-3p, and miR-125-3p expression were significantly downregulated compared to the unstimulated control (Figure 2B). These four miRs were further selected for 48 h HG stimulation experiments. When HAECs were stimulated with HG for 48 h, the expression levels of miR-146a-5p, miR-339-5p, and miR-874-3p were significantly downregulated compared to the unstimulated control (Figure 2C).

The miR target analysis showed homologies between the 3′-UTR of the human IRAK-1 mRNA and miR-146a-5p (two binding sites), miR-339-5p, and miR-874-3p, indicating potential regulation of IRAK-1 (Figure 3A). We performed a miR mimic competitive transfection assay to determine whether mimics of these three miRs could decrease IRAK-1 gene expression. As shown in Figure 3B, only miR-146a-5p transfection significantly decreased IRAK-1 gene expression in the 48 h HG-stimulated HAECs. The osmotic control experiments showed that mannitol treatment did not modulate the expression levels of miR-146a-5p (Supplemental Data 3). To investigate whether miR-146a-5p can interact with the IRAK-1 mRNA 3′-UTR, a luciferase reporter assay was performed. As shown in Figure 3C, cotransfection of pGL3-IRAK-1-3′-UTR and the miR-146a-5p mimic resulted in a decrease in luciferase signal to 65% of that in the negative control, which confirmed direct binding of miR-146a-5p to the IRAK-1 3′-UTR. Transfection of the miR-146a-5p mimic also significantly attenuated IRAK-1 protein expression in HG-stimulated HAECs (Figure 3D). By contrast, the stimulatory effects of HG on IRAK-1 expression were potentiated by a miR-146a-5p inhibitor (Figure 3E). To determine whether reduced IRAK-1 expression was associated with reduced endothelial inflammation in miR-146a-5p mimic-transfected, HG-stimulated HAECs, we measured VCAM-1 and ICAM-1 expression levels and THP-1 adhesion. As anticipated, both VCAM-1 and ICAM-1 levels were attenuated by transfection of the miR-146a mimic (Figure 3F). Furthermore, THP-1 adhesion to HAECs was also significantly reduced (Figure 3G).

To determine whether enhanced IRAK-1 expression was associated with HG-induced endothelial inflammation, an IRAK-1 siRNA transfection experiment was performed. After IRAK-1 siRNA transfection, both IRAK-1 mRNA (Figure 4A) and protein levels (Figure 4B) were significantly downregulated in HG-stimulated HAECs. Furthermore, similar to the effects of the miR-146a mimic, IRAK-1 siRNA significantly inhibited the expression of HG-stimulated VCAM-1/ICAM-1 gene expression (Figure 4C) and THP-1 adhesion to HAECs (Figure 4D), indicating that HG-induced endothelial inflammation was mediated partially through IRAK-1.

To examine the effect of the miR-146a-5p mimic on the expression of endothelial IRAK-1 and ICAM-1 in vivo, we performed IHC on aortic tissues from db/db type 2 diabetic mice. As shown in Figure 5A, aortic endothelial IRAK-1 and ICAM-1 protein levels from miR-146a-5p mimic-treated mice were dramatically decreased, as compared to negative control or vehicle-treated mice. This demonstrates that miR-146a-5p may have therapeutic potential, mitigating endothelial dysfunction through the downregulation of both IRAK-1 and ICAM-1. The proposed role of miR-146a-5p in regulating HG-induced endothelial inflammation via IRAK-1 is shown in Figure 5B.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.