MicroRNA-203 mimics age-related aortic smooth muscle dysfunction of cytoskeletal pathways

Abstract

Increased aortic stiffness is a biomarker for subsequent adverse cardiovascular events. We have previously reported that vascular smooth muscle Src-dependent cytoskeletal remodelling, which contributes to aortic plasticity, is impaired with ageing. Here, we use a multi-scale approach to determine the molecular mechanisms behind defective Src-dependent signalling in an aged C57BL/6 male mouse model. Increased aortic stiffness, as measured in vivo by pulse wave velocity, was found to have a comparable time course to that in humans. Bioinformatic analyses predicted several miRs to regulate Src-dependent cytoskeletal remodelling. qRT-PCR was used to determine the relative levels of predicted miRs in aortas and, notably, the expression of miR-203 increased almost twofold in aged aorta. Increased miR-203 expression was associated with a decrease in both mRNA and protein expression of Src, caveolin-1 and paxillin in aged aorta. Probing with phospho-specific antibodies confirmed that overexpression of miR-203 significantly attenuated Src and extracellular signal regulated kinase (ERK) signalling, which we have previously found to regulate vascular smooth muscle stiffness. In addition, transfection of miR-203 into aortic tissue from young mice increased phenylephrine-induced aortic stiffness ex vivo, mimicking the aged phenotype. Upstream of miR-203, we found that DNA methyltransferases (DNMT) 1, 3a, and 3b are also significantly decreased in the aged mouse aorta and that DNMT inhibition significantly increases miR-203 expression. Thus, the age-induced increase in miR-203 may be caused by epigenetic promoter hypomethylation in the aorta. These findings indicate that miR-203 promotes a re-programming of Src/ERK signalling pathways in vascular smooth muscle, impairing the regulation of stiffness in aged aorta.

Keywords: aortic stiffness; cytoskeleton; focal adhesion; microRNA; vascular smooth muscle.

Figure 1

Aortic stiffness is increased in aged mice. Aortic stiffness assessed in vivo by pulse wave velocity (PWV). (A) Blood flow waveforms were continuously recorded for 20 sec. at a proximal and a distal location along the aorta with simultaneous ECG. PWV was calculated by dividing the distance between the proximal and distal locations by the difference between the proximal and distal transit times and expressed in mm/msec. (B) PWV was measured in 4‐ (n = 11), 10‐ (n = 9), 18‐ (n = 8) and 24‐month‐old (n = 7) male mice to obtain a time course of aortic stiffness development. P < 0.05 compared to 4 months (*) and 10 months (†) (two‐tailed Student's t‐test).

Figure 2

Ageing alters the expression of actin cytoskeletal‐ and focal adhesion‐regulating microRNAs in the mouse thoracic aorta. (A) TargetScan and miRbase were used to identify miRs that could potentially regulate expression of genes involved in FA and ERK signalling pathways, by reverse target prediction (i.e. using the gene targets to select the miRs of interest). (B) The expression level of miRs identified in the above table were compared from young (3 months) and aged (24–29 months) mice (n = 12 mice). *P < 0.05 (two‐tailed Student's t‐test).

Figure 3

Overexpression of miR‐203 decreases both mRNA and protein levels of predicted targets in A7r5 cells. (A) Schematic of the protocol for transfection of A7r5 cells with miR‐203 mimic or control mimic. (B) mRNA expression levels for Cav‐1, Crk, PKC‐α, Pxn, Src and Tln2 compared in cells transfected with miR‐203 mimic or control (n = 6 experiments). (C) (Top) Representative western blots depicting Src, Cav‐1 and paxillin expression in control and miR‐203 mimic transfected cells. (Bottom) Normalized expression of Src, Cav‐1, and paxillin (n = 12 experiments). *P < 0.05 (two‐tailed Student's t‐test).

Figure 4

Ageing regulates the expression of miR‐203 targets in mouse aorta. mRNA and protein expression of the validated miR‐203 targets were analysed to determine if ageing regulated their expression in the thoracic aorta. (A) mRNA expression levels of Src, Cav‐1 and Pxn compared in the thoracic aorta from young and aged mice (n = 10–11 mice). (B) XY scatter plots generated to compare the levels of miR‐203 with the mRNA levels of its targets in the aorta from each individual mouse. (C) Typical western blots depicting Src, Cav‐1 and paxillin protein expression in young and aged aorta are shown on the left, with the subsequent quantification comparing their normalized expressions on the right (n = 8 mice). *P < 0.05 (two‐tailed Student's t‐test).

Figure 5

miR‐203 overexpression downregulates agonist‐induced FA and ERK signalling. A7r5 cells were stimulated with DPBA (3 μM for 10 min.) 72 hrs post‐transfection to determine the influence of miR‐203 on signalling pathways downstream of Src. (A) Typical western blots depicting FA signalling in cells transfected with miR‐203 or control mimic. DPBA‐induced phosphorylation of FAK pY‐925 (B), CAS pY‐165 (C), paxillin pY‐118 (D), p‐ERK1 (E) and p‐ERK2 (F) compared in A7r5 cells (n = 7 experiments) transfected with miR‐203 or control mimic. P < 0.05 compared to vehicle control (*) and negative miR mimic control (#) (two‐way anova).

Figure 6

Overexpression of miR‐203 in young aorta reproduces the ageing phenotype of increased aortic stiffness. (A) Schematic of the protocol for transfection of aortic tissue with miR‐203 mimic or control mimic. (B) miR‐203 expression levels in tissue transfected with miR‐203 mimic or control (n = 4 experiments). (C) Agonist‐induced stiffness increase in young (white, n = 10) versus aged (black, n = 6) aorta and young aorta transfected with scrambled miR mimic control (light grey, n = 10) versus miR‐203 mimic (pattern grey, n = 9). *P < 0.05 from young aorta or # scrambled mimic control (two‐tailed Student's t‐test).

Figure 7

DNA methyltransferases (DNMTs) influence miR‐203 expression. (A) Gene expression of DNMT1, 3a and 3b in young (white) and aged (black) proximal mouse aorta (n = 10 mice). *Significant difference from young mice (two‐tailed Student's t‐test). (B) miR‐203 expression in cells treated with the DNMT inhibitor 5‐Aza for 72 hrs and harvested for quantification of miR‐203 expression (n = 6 experiments). *P < 0.05 (two‐tailed Student's t‐test).

Figure 8

Proposed mechanism. Ageing triggers an increase in miR‐203 expression, leading to a reduction in Src‐mediated signalling. The age‐related increase in miR‐203 expression, at least partially caused by hypomethylation of the miR‐203 promoter region, negatively influences the expression of Src, Cav‐1 and paxillin. Consequently; downstream tyrosine phosphorylation (p‐Tyr) of FAK, paxillin and CAS and ERK activation are impaired, contributing to the age‐related defect in FA remodelling and impaired regulation of stiffness.