microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression

Figures

• 

  Figure 1. Dietary CLA blend decreased aortic miR-155 expression in ApoE−/− mice. ApoE−/− mice were fed a ND or a 1% CD for 12 weeks or a 1% CD for 12 weeks with the final 4 weeks supplemented with 1% CLA blend (1% CD + CLA). Aortae were dissected and RNA was isolated. Previously, transcriptomic analysis was performed on murine aortae from the 1% CD + CLA group and the 1% CD group by McCarthy et al. (A) In silico, IPA analysis was performed to identify upstream regulators of the CLA-responsive gene set (n = 1082; FDR p < 0.05; Fold-change >1.7). The activation Z-score (≥ 2 or ≤ −2) and upstream miRNA regulators identified were graphed, the overlap p-value was set to p < 0.05. (B) miR-155 expression was analyzed by qRT-PCR using RNU6B as the reference gene. (C) BCL-6 and (D) SHIP-1 expression analyzed by qRT-PCR using 18S as the reference gene and graphed as fold over control (F.O.C.). qRT-PCR results are expressed as mean ± SEM (4 ≤ n ≤ 9). Statistical analysis was performed using a Mann-Whitney U test. *p < 0.05 was considered statistically significant. * over the columns are comparisons with the ND, control group. Significance between other groups is indicated by capped lines.

• 

  Figure 2. Polarization of THP-1 macrophages. THP-1 monocytes were polarized into THP-1 macrophages (M0) using PMA (320 nmol/L) for 72 h, after which cells underwent a 24 h rest phase to allow the M0 phenotype to develop. Macrophages were then treated for 48 h with 100 ng/ml LPS and 20 ng/ml IFN-γ to polarize cells to M1 macrophages and 20 ng/ml IL-4 and IL-13 for M2 macrophages. The supernatants were analyzed by ELISA for (A) IL-1β, (B) TNF-α, and (C) IL-10 secretion. RNA was analyzed for (D) MR expression by qRT-PCR using 18S rRNA (18S) as the reference gene and graphed as fold over control (F.O.C.). Flow cytometry analysis was used to determine the median fluorescence intensity of (E) HLA-DR (FITC) and (F) MR (APC) and illustrated using a representative histogram plot. (G) Quadrant regions were used to detect the percentage of macrophages positive for HLA-DR (FITC) and MR (APC) following the respective treatments. The percentage of (H) HLA-DR and (I) MR positive cells was determined and graphed. Error bars are representative of independent experiments performed in duplicate for TNF-α (n = 3), IL-10 (n = 4), IL-1β (n = 7), MR (n = 3) and HLA-DR (n = 3). Statistical analysis was carried…

• 

  Figure 3. CLA decreased miR-155 and increased protein expression of miR-155 targets, BCL-6 and SHIP-1, in polarized M2 THP-1 macrophages. Polarized THP-1 macrophages were treated with CLA blend (25 µmol/L) for 18 h using DMSO (0.1%) as the control. microRNA was converted to cDNA which then underwent qRT-PCR analysis for (A) miR-155 expression using RNU48 and RNU6B as the reference genes. Error bars are representative of four independent experiments (n = 4) carried out in duplicate and averaged and graphed as fold over control (F.O.C.). mRNA was analyzed by qRT-PCR for (B) IL-10 expression (n = 5). Western blotting and densitometry analysis were performed on (C) BCL-6 (n = 4), (D) SHIP-1 (n = 5), (E) p-STAT-3 (n = 4), and (F) MR (n = 4) using β-Actin and GAPDH as controls. Statistical analysis was carried out using multiple paired t-tests where *p < 0.05 represent statistical significance. * over the columns represents statistical significance when compared to the control group M0 treated with DMSO. Significance between other groups is indicated by capped lines.

• 

  Figure 4. p-STAT-3, BCL-6, and BCL-2 protein expression is decreased following miR-155 mimic transfection of THP-1 M0 macrophages. (A) Light microscopy images of THP-1 monocytes which were differentiated to macrophages using PMA (320 nmol/L) for 72 h followed by a 24-h rest phase. Macrophages were transfected for 24 h with 20nmol/L of miR-155 mimic using Lipofectamine™ 3000. Scrambled negative control mimic and Lipofectamine™ 3000 only (untreated) were used as controls. The media was removed, and cells were stimulated with LPS (100 ng/ml) for 18 h using dH2O as the control. Cells were harvested for RNA and protein. Transfection efficacy was analyzed for (B) miR-155 expression (n = 5) by qRT-PCR and graphed as fold over control (F.O.C.). Western blotting and densitometry analysis were performed on (C) p-STAT-3 (n = 4), (D) Total STAT-3 (n = 3), (E) p-STAT-1 (n = 3), (F) SHIP-1 (n = 5), (G) BCL-6 (n = 5), and (H) BCL-2 (n = 3) expression, using β-actin and GAPDH as the loading controls. Statistical analysis was performed using multiple paired t-tests where *p < 0.05 and **p < 0.01 represented statistical significance. * over the columns represent statistical significance when compared to the…

• 

  Figure 5. Patients with unstable CAD have a decreased concentration of uEVs. EVs were isolated from 6 ml of urine using a benchtop centrifugation protocol. (A) Images of uEVs were taken using an FEI Tecnai 12 Transmission Electron Microscope (Magnification: 43,000× and 135,000×). (B) The total concentration of particles (5-200nm) was determined by NTA using the NanoSight NS300. (C) R Studio analysis was performed to compare the size distribution and concentration of the NTA data. (D) The EV (Bodipy+) concentration was determined by flow cytometry using the ImageStream™X Mark II. (E) The percentage of CD63+ Bodipy+ EVs was also determined. (F) Images of EVs stained with Bodipy and positive for the fluorescent antibodies CD63 and CD45 were obtained using the ImageStream™X Flow Cytometer. Statistical analysis was performed using nonparametric Mann Whitney U tests where *p < 0.05 and **p < 0.01 represent statistical significance. Statistical significance between groups is indicated by capped lines. ns, not significant.

• 

  Figure 6. Patients with unstable CAD have increased uEV miR-155 expression and alterations in CD45+, CD11b+, and CD16+ uEVs. For qRT-PCR analysis, four EV pellets (derived from 6-ml urine) were re-suspended in TRIzol™ for RNA extraction. 100ng of RNA was reverse transcribed to cDNA and qRT-PCR analysis was performed for (A) miR-155 (n = 11 per group) and (B) miR-21 (5 stable and 9 unstable CAD patients) expression using the reference genes, RNU6B and RNU48. For flow cytometry analysis, four pellets were re-suspended in 300 μl of sterile-filtered PBS, 50 µl of each sample was stained with the membrane stain, Bodipy and the antibodies CD45, CD11b, CD14, and CD16 (n = 1 per group). Ten thousand EVs positive for Bodipy were analyzed. (C) The percentage of CD45 and CD11b fluorescence antibody relative to Bodipy stain was analyzed. (D) The concentration of CD45 and CD11b fluorescent EVs per ml was analyzed. (E) The percentage of CD14 and CD16 fluorescence antibody relative to Bodipy stain was analyzed. (F) The concentration of CD14 and CD16 fluorescent EVs per ml was analyzed. Statistical analysis was performed using non-parametric Mann-Whitney U tests where *p < 0.05, **p < 0.01, and ***p <…