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.
Highlights
Cardiovascular diseases are the leading cause of death worldwide [1]
In this study we demonstrate, for the first time, that age-related increases in miR-203 expression in mouse aorta are associated with downregulation of critical focal adhesion (FA) signalling proteins in the Vascular smooth muscle cell (VSMC), providing a mechanistic basis for the previously described defect in FA signalling with aortic ageing [2]
That miR-203 contributes to the impairment of FA signalling in aged aorta, which is thought to be critical in the normal functioning of the proximal aorta as a buffer against the high systolic pressures generated by the heart
Summary
Cardiovascular diseases are the leading cause of death worldwide [1]. In the vascular system, the proximal aorta plays a critical role as a shock absorber against the intermittent pulsatile output from the heart, and provides a steady flow to smaller vessels downstream [2, 3]. Vascular smooth muscle cell (VSMC) contraction is predominantly initiated by Ca2+-dependent activation of myosin light chain kinase, which phosphorylates the myosin light chains, leading to formation of actomyosin cross-bridges and increased VSMC stiffness [9]. We sought to determine whether aberrant expression of miRs could play a role in the age-related impairment of Src-mediated plasticity of aortic smooth muscle. We report here that age-related upregulation of miR-203, in a cause-and-effect manner, impairs key phosphorylation events downstream of Src tyrosine kinase, which is associated with increased tissue stiffness. Tissues were quick-frozen in an acetone-dry ice slurry containing 10 mM dithiothreitol (for RNA-based studies) or 10 mM dithiothreitol and 10% trichloroacetic acid (for western blot studies), as described previously [43]
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