miR-133a Replacement Attenuates Thoracic Aortic Aneurysm in Mice

Abstract

Background Thoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR-133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR-133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model. Methods and Results TAA was induced in mice. Copy number of miR-133a was reduced in TAA tissue and linear regression analysis confirmed an inverse correlation between aortic diameter and miR-133a. Analyses of phenotypic markers revealed an mRNA expression profile consistent with myofibroblasts in TAA tissue. Fibroblasts were isolated from the thoracic aortae of mice with/without TAA. When compared with controls, miR-133a was reduced, migration was increased, adhesion was reduced, and the ability to contract a collagen disk was increased. Overexpression/knockdown of miR-133a controlled these phenotypes. After TAA induction in mice, a single tail-vein injection of either miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Overexpression of miR-133a attenuated TAA development. The pro-protein convertase furin was confirmed to be a target of miR-133a by luciferase reporter assay. Furin was elevated in this murine model of TAA and repressed by miR-133a replacement in vivo resulting in reduced proteolytic activation. Conclusions miR-133a regulates aortic fibroblast phenotype and over-expression prevented the development of TAA in a murine model. These findings suggest that stable alterations in aortic fibroblasts are associated with development of TAA and regulation by miR-133a may lead to a novel therapeutic strategy.

Keywords: fibroblast; furin; miR‐133a; myofibroblast; thoracic aortic aneurysm.

Figure 1. MiR‐133a is reduced in thoracic aortic aneurysm tissue.

A, Percentage change in aortic diameter at 4 weeks (59.50±4.90%, n=9), 8 weeks (64.25±6.77%, n=10), and 16 weeks (62.89±6.75%; n=8) after aneurysm induction when compared with baseline diameter measurements. *One‐way ANOVA (Dunn's method) found differences between 4 weeks (P<0.001), 8 weeks (P<0.001), and 16 weeks (P<0.001) after aneurysm induction vs baseline measurements. B, MiR‐133a copy number in aortic tissue for sham‐operated, nonaneurysmal control (0.74±0.099 copies per U6; n=10) and 4 weeks (0.45±0.082 copies per U6; P=0.623; n=7), 8 weeks (0.19±0.073 copies per U6; n=7), and 16 weeks (0.053±0.016 copies per U6; n=3) after aneurysm induction. *Kruskal‐Wallis 1‐way ANOVA on ranks found difference between 8 (P=0.004) and 16 (P=0.002) weeks vs control. Additionally, in a 1‐way ANOVA all pairwise multiple comparison procedure (Tukey test), difference was detected between control vs 16 weeks (P=0.004) and control vs 8 weeks (P=0.008). C, Linear regression analysis demonstrating inverse correlation between aortic diameter (mm) and miR‐133a copy number in aortic tissue (n=34; r=−0.6356; P<0.05). In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median.

Figure 2. MiR‐133a was decreased in fibroblasts isolated from thoracic aortic aneurysm (TAA) tissue.

A, Thoracic aortic diameter of sham‐operated control (701.22±17.24 µm) and TAA (1019.49±41.82 µm) 4 weeks after induction surgery (n=10). *Student t test and Welch's t test found a difference between TAA vs control (P<0.001). B, Fold change in smooth muscle myosin (Myh11) mRNA copy number per DDR2 copy number in control (1±0.10‐fold) and TAA (2.71±0.67‐fold) aortic tissue, as well as, alpha smooth muscle actin (α‐SMA) copy number in control (1±0.20‐fold) and TAA (1.24±0.18‐fold) tissue (n=10). *Mann‐Whitney rank‐sum test and Student t test found a difference between TAA vs control Myh11 copy number (P=0.045). Student t test and Welch's t test did not find a difference between TAA α‐SMA vs control (P=0.378). C, Detection of cell type–specific mRNAs in whole aortic tissue, and isolated fibroblasts from control and TAA aortae. Mesenchymal cell specific vimentin, smooth muscle cell–specific desmin, endothelial specific von Willebrand factor (vWF), and fibroblast specific DDR2 were all detected in whole aortic tissue homogenate, while only vimentin and discoidin domain receptor family, member 2 (DDR2) were present in the isolated control and TAA fibroblasts. No statistical test was performed. This is an analysis of presence or absence of cell specific mRNAs by droplet digital polymerase chain reaction (ddPCR) (n=10). D, Fold change in Myh11 copy number in control (1±0.28‐fold) and TAA (4.47±1.23‐fold) fibroblasts and also α‐SMA copy number in control (1±0.187‐fold) and TAA (1.003±0.26‐fold) fibroblasts (n=6). ^*Student t test and Welch's t test found a difference between TAA and control Myh11 copy number (P=0.036); however, no difference was found between TAA and control α‐SMA copy number (P=0.992). E, MiR‐133a copy number per U6 in control (1.72±0.46‐fold) and TAA (0.41±0.12‐fold) fibroblasts (n=10). *Mann‐Whitney rank‐sum test found a difference between TAA vs control aortic fibroblasts (P=0.007). F, Pri‐miR‐133a‐1 copy number per GAPDH in control (0.018±0.00259 copy per U6) and TAA (0.016±0.00224 copy per U6) fibroblasts (n=6). Student t test and Welch's t test found no difference between control vs TAA fibroblasts (P=0.516). G, Pri‐miR‐133a‐2 copy number per GAPDH in control (2.40e‐3±9.56e‐4) and TAA (9.75e‐4±2.83e‐4) fibroblasts (n=6). Student t test and Welch's t test found no differences between control vs TAA fibroblasts (P=0.205). In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median. In the vertical multibar graphs, the mean is represented by the bar, and the upper standard error of the mean is represented by the error bar.

Figure 3. Phenotype was altered in fibroblasts isolated from thoracic aortic aneurysm (TAA) tissue.

A, Migration of aortic fibroblasts as determined by transwell assay. Displayed are the percent of total cells migrated through an 8‐µm porous membrane following 24‐hour incubation of control (0.47±0.038%, n=10) and TAA (0.62±0.041%, n=9) fibroblasts. *Student t test and Welch's t test found differences between TAA vs control fibroblasts (P=0.013). B, Adhesion of aortic fibroblasts following repeat washes in an automated plate washer. Displayed is a fold change in cell count compared with unwashed in control (−0.77±0.022‐fold) and TAA (−0.85±0.018‐fold) fibroblasts (n=10). *Student t test and Welch's t test found a difference between 4‐week TAA vs control fibroblasts (P=0.009). C, Fold change in contraction rate of a collagen disk seeded with control (1.00±0.39‐fold, n=12) or TAA (1.38±0.34‐fold, n=8) fibroblasts. *Student t test and Welch's t test found a difference in contraction rate between control and TAA fibroblasts (P=0.034). D, Representative images of collagen disks seeded with control or TAA fibroblasts over 7 hours (images taken at 1, 3, 5, 6, and 7 hours from collagen polymerization). In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median.

Figure 4. MiR‐133a modulates aortic fibroblast phenotype.

A, Fold change in control (green) and TAA (red) fibroblast miR‐133a copy number following transfection with the negative control microRNA mimic (Neg. Ctrl.) (control: 1.00±0.35‐fold; TAA: 1.00±0.15‐fold; n=8), miR‐133a mimic (control: 5637.49±3247.71‐fold, P<0.001, n=8; TAA: 3905.97±1570.75‐fold, P<0.001, n=7), and anti‐miR‐133a oligonucleotide (control: 1.75±0.57‐fold, P=0.495; TAA: 2.09±0.37‐fold, P=0.421; n=8). *, # Kruskal‐Wallis 1‐way ANOVA on ranks with Tukey test for post hoc analysis found differences between miR‐133a copy number in both control and TAA fibroblasts transfected with the miR‐133a mimic vs fibroblasts transfected with the negative control mimic. B, Percentage of total control fibroblasts migrated through an 8µm porous membrane following transfection with the negative control microRNA mimic (0.52±0.017%), miR‐133a mimic (0.49±0.010%), and anti‐miR‐133a oligonucleotide (0.58±0.016%) (n=8). *One‐way ANOVA, multiple comparisons vs control group (Bonferroni t test) found a difference between anti–miR‐133a oligonucleotide vs negative control mimic (P<0.012). ^#One‐way ANOVA, all pairwise multiple comparison procedure (Tukey test) found a difference between anti–miR‐133a vs miR‐133a (P<0.001). C, Percent of total TAA fibroblasts migrated through an 8‐µm porous membrane following transfection with the negative control microRNA mimic (0.56±0.034%), miR‐133a mimic (0.40±0.026%), and anti–miR‐133a oligonucleotide (0.63±0.043%) (n=8). *One‐way ANOVA, multiple comparisons vs control group (Bonferroni t test) found difference between miR‐133a mimic vs negative control mimic (P=0.006). ^#One‐way ANOVA, multiple comparison procedure (Tukey test) vs miR‐133a mimic found difference between anti–miR‐133a vs miR‐133a (P<0.001). D, Percent change in control fibroblast adhesion following transfection with the negative control mimic (0.00±0.7%), miR‐133a mimic (3.4±1.0%), and anti–miR‐133a oligonucleotide (1.3±0.08%) (n=12). *One‐way ANOVA, multiple comparisons vs control group (Bonferroni t test) found difference between miR‐133a mimic vs negative control mimic (P=0.016). E, Percentage change in TAA fibroblast adhesion following transfection with the negative control mimic (0.00±0.009%), miR‐133a mimic (7.3±0.08%), and anti–miR‐133a oligonucleotide (−9.0±0.17) (n=12). *One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found differences between miR‐133a vs negative control (P=0.016) and anti–miR‐133a vs negative control (P=0.016). ^#One‐way ANOVA, multiple comparisons vs miR‐133a group (Dunn's method) found difference between anti–miR‐133a vs miR‐133a (P<0.001). F, Fold change (F/C) in contraction rate of a collagen disk seeded with control fibroblasts transfected with negative control mimic (1.0±0.049, n=12), miR‐133a mimic (0.30±0.042, n=10), and anti–miR‐133a oligonucleotide (1.18±0.14, n=12). *One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found differences between miR‐133a vs negative control (P=0.006). ^#One‐way ANOVA, multiple comparisons vs miR‐133a group (Dunn's method) found differences between anti–miR‐133a vs miR‐133a (P<0.001). G, F/C in contraction rate of a collagen disk seeded with TAA fibroblasts transfected with negative control mimic (1.00±0.033, n=12), miR‐133a mimic (0.32±0.058, n=10), and anti–miR‐133a oligonucleotide (1.59±0.11; n=12). *One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found differences between anti–miR‐133a vs negative control (P=0.021) and between miR‐133a vs negative control (P=0.007). ^#One‐way ANOVA, multiple comparisons vs miR‐133a group (Dunn's method) found difference found between miR‐133a vs negative control (P<0.001). In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median. In D through G, the dashed line represents referent control levels.

Figure 5. MiR‐133a attenuates the development of thoracic aortic aneurysms (TAA) in mice.

A, Fold change in aortic tissue miR‐133a copy number following TAA induction in mice transfected with either the control virus (1.00±0.16‐fold) or the miR‐133a over expression virus (1.81±0.34‐fold) compared with nontransfected mice with TAA (red dashed line) (n=14). *Student t test and Welch's t test found a difference between miR‐133a virus vs control virus (P=0.048). B, Percentage change in aortic diameter in nontransfected (no virus) and transfected mice with the control virus, and miR‐133a virus 4 weeks (no virus: 55.70±5.80%, n=9; control virus: 65.95±1.97%, n=9; miR‐133a virus: 24.64±2.19%, n=9), 8 weeks (no virus: 0.64±0.07%, n=10; control virus: 0.49±0.03%, n=9; miR‐133a virus: 0.23±0.040%, n=6), and 16 weeks (no virus: 0.63±0.07%, n=8; control virus: 0.62±0.08%, n=7; miR‐133a virus: 0.25±0.05%, n=10) after aneurysm induction. *One‐way ANOVA, multiple comparisons vs control group (Bonferroni t test) found difference between miR‐133a virus vs no virus at 4 (P<0.001) and 16 (P<0.001) weeks. No difference was found between control virus vs no virus at 4 (P=0.359) or 16 (P=1) weeks. One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found difference between miR‐133a virus vs no virus at 8 (P<0.001) weeks. No difference was found between control virus vs no virus at 8 weeks (P=0.761). ^#One‐way ANOVA, all pairwise multiple comparison procedure (Tukey test) found differences between miR‐133a virus vs control virus at 4 weeks (P<0.001) and 16 weeks (P=0.001), and no difference between no virus vs control virus at 4 (P=0.366) or 16 (P=0.999) weeks. One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found a difference between miR‐133a virus vs control virus at 8 weeks (P=0.02) and no difference between no virus vs control virus at 8 weeks (P=1). C, Immunofluorescence (top) and modified Verhoeff–Van Gieson stain elastic stain (bottom) of non‐TAA control and nontransfected (no virus) and transfected mice with the control virus, and miR‐133a virus aortic sections 16 weeks following the TAA induction procedure. In the immunofluorescent images, elastin is seen as green autofluorescence, nuclei are stained blue with DAPI, and red is a primary antibody to the green fluorescent protein conjugated with a secondary antibody labeled with a red Alexaflour. White arrows indicate transduced cells and elastin breaks. In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median. D, Fold change in aortic tissue Myh11 copy number per DDR2 copy following TAA induction in mice transfected with either the control virus (0.79±0.15‐fold) or the miR‐133a overexpression virus (0.33±0.07‐fold) compared with nontransfected mice with TAA (red dashed line) (n=11). *Student t test and Welch's t test found a difference between miR‐133a virus vs control virus (P=0.018).

Figure 6. MiR‐133a directly regulates furin translation.

A, Sequence alignment between the mature miR‐133a transcript (accession: MIMAT0000427) with the conserved sequence region of the furin 3′ untranslated region (UTR). Transcript in uppercase is conserved in at least 42 species, and transcript in lowercase is conserved in at least 21 species (TargetScan 7.2). Below is the gene mutation sequence introduced in the 3′UTR of the mutant furin reporter plasmid for confirmation of direct targeting. B, Furin is elevated in thoracic aortic aneurysm (TAA) fibroblasts. Displayed is the fold change in furin abundance of control (1±0.05‐fold, n=15) and TAA (1.89±0.20‐fold, P<0.001, n=12) fibroblasts with representative immunoblot above. *Mann‐Whitney rank‐sum test found difference between TAA vs control (P<0.001). C, MiR‐133a directly targets the GACCA consensus sequence located in the furin 3′UTR. Displayed is the fold change in luminescence following cotransfection of HT1080 cells with the following 3′UTR luciferase reporter constructs and either a nontargeting negative control microRNA mimic or miR‐133a: the wild‐type furin 3′UTR construct cotransfected with either the negative control (1.00±0.10‐fold) or miR‐133a mimic (0.62±0.10‐fold); the mutated furin 3′UTR (with GACCA replace with CUGGU by site‐directed mutagenesis) construct cotransfected with the negative control (1.00±0.03‐fold) and miR‐133a (1.12±0.11); a random sequence 3′UTR construct (confirmed to not contain GACCA) with the negative control (1.00±0.09) and miR‐133a (1.14±0.09), and a positive control construct (which contains no 3′UTR [no UTR]) cotransfected with the negative control (1.00±0.14‐fold) or miR‐133a (1.22±0.09‐fold) (n=5). *Student t test and Welch's t test found differences in luminescence between the furin 3′UTR transfected with the miR‐133a mimic vs when transfected with the nontargeting, negative control, microRNA mimic (P=0.024). D, Furin abundance in control fibroblasts following transfection with the negative control microRNA mimic (1.0±0.14; n=8), miR‐133a mimic (0.609±0.070; P<0.399; n=6), and anti–miR‐133a oligonucleotide (1.59±0.22; n=6) with representative immunoblot above. *One‐way ANOVA, multiple comparisons vs control group (Bonferroni t test) found difference between anti–miR‐133a vs negative control (P=0.041). ^#One‐way ANOVA, all pairwise multiple comparison procedure (Tukey test) found difference between anti–miR‐133a vs miR‐133a (P=0.006). E, Furin abundance in TAA fibroblasts following transfection with the negative control microRNA mimic (1.00±0.116; n=9), miR‐133a mimic (0.609±0.070; P=0.041; n=9), and anti–miR‐133a oligonucleotide (1.16±0.11; n=9) with representative immunoblot above. *One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found difference between miR‐133a and negative control group (P=0.054). ^#One‐way ANOVA, multiple comparisons vs miR‐133a group (Dunn's method) found difference between anti–miR‐133a vs miR‐133a (P=0.004). In D, the bar height represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median. In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median.

Figure 7. MiR‐133a overexpression modulates furin in aortic tissue.

A, Proposed pathway of miR‐133a regulation of extracellular matrix (ECM) remodeling. B, representative western blot and fold change in furin abundance in control tissue (1±0.055, n=12) and thoracic aortic aneurysms (TAA​) tissue transfected with no virus (2.5±0.31, n=12), control virus (2.54±0.15, n=12), and miR‐133a virus (1.18±0.15, P=1, n=9). *One‐way ANOVA, multiple comparisons vs control group (Dunn's method) found differences between control group vs no virus (P<0.001) and vs control virus (P<0.001). ^#One‐way ANOVA, multiple comparisons vs miR‐133a group (Dunn's method) found differences between miR‐133a vs control virus (P=0.002). C, representative western blot and fold change in membrane type 1 matrix metalloproteinase (MT1‐MMP) abundance in control tissue (1.03±0.059, n=11) and TAA tissue transfected with no virus (1.38±0.11; n=12), control virus (1.24±0.040; n=12), and miR‐133a virus (0.94±0.089; n=12). *One‐way ANOVA multiple comparisons vs control group (Bonferroni t test) found difference between no virus vs control (P=0.005) and control virus vs control (P=0.040). ^#One‐way ANOVA, all pairwise multiple comparison procedure (Tukey test) found difference between no virus vs miR‐133a virus (P=0.002) and control virus vs miR‐133a virus (P=0.041). In the vertical point plots, the solid line represents the mean, the upper and lower bars represent the standard error of the mean, and the dotted line represents the median.