Today's open access paper is a discussion of microRNA regulation of cell behavior in the context of age-related arterial stiffness. The stiffening of blood vessels with age is an important issue, as it produces hypertension via disruption of the feedback mechanisms that control blood pressure. The raised blood pressure of hypertension causes structural damage throughout the body, increasing mortality and accelerating progression of age-related disease.
Every distinct form of organ or tissue dysfunction in aging is made of up many layers. At the base are fundamental causes, forms of damage that arise from the normal operation of metabolism and have no deeper origin. In the case of arterial stiffness that includes loss of elastin and persistent cross-links in the extracellular matrix, both of which reduce the elasticity of blood vessel wall tissues. Some of us tend to put senescent cell accumulation in that layer as well, though the growing number of senescent cells with age is likely to be largely a result of immune system aging. The pro-inflammatory signaling of senescent cells is implicated in smooth muscle dysfunction in blood vessel walls.
These forms of damage feed into the next layer of tissue dysfunction, in which regulatory systems that control gene expression change in response to that damage. These include microRNAs, transcription factors, and other mechanisms involved in the control of the expression of networks of genes in the cell. Some responses are adaptive and some are maladaptive. Altered regulation then produces the uppermost layer of tissue dysfunction, consisting of broad and problematic changes in cell behavior and cell signaling, driven by alterations in protein abundance within the cell and changes in the environment of signals that pass between cells. The vast majority of research into aging and age-related disease focuses on these upper layers: gene expression and its regulation in the aged tissue environment. All too little work is focused on deeper causes.
Large artery stiffness (LAS) is a major, independent risk factor underlying cardiovascular disease that increases with aging. Arterial stiffness is determined by the composition and organization of the extracellular matrix, as well as the cytoskeletal properties of vascular smooth muscle cells. Therefore, investigators have concentrated on defining the biochemical and associated structural changes responsible for increased arterial stiffness, as well as the upstream regulatory pathways driving these changes. From this perspective, the microRNA system has emerged as a potential culprit, since this highly versatile signaling pathway can coordinate cellular adaptations in response to developmental and environmental signals and has been found to play key roles in vascular smooth muscle development and function. However, since each cell contains hundreds of distinct microRNAs, and these microRNA profiles differ across cell types, identifying candidate microRNAs involved in regulating arterial stiffness poses a difficult challenge.
Initial clues implicating dysregulation of specific microRNAs in LAS emerged from two approaches. As patients with LAS can be identified by measuring pulse wave velocity (PWV) using non-invasive techniques, investigators looked for alterations in microRNA signaling associated with elevated PWV. This approach yielded identification of two microRNAs linked to elevated PWV: miR-765 and miR-1185. The second approach was based on the epidemiological observation that the prevalence of LAS increases markedly with aging. Furthermore, mice also display aging-associated increases in PWV that model LAS. Thus, investigators checked for microRNAs that show altered expression with aging in blood samples from humans and in mouse aorta. This approach led to the identification of several candidate microRNAs that show altered expression with aging: mir-29, miR-34a, miR-92a, miR-137, miR-181b, miR-203, and miR-222. Mir-29, miR-34a, miR-137, miR-203, and miR-222 increase with aging in mouse aorta and miR-34a has been shown, very recently, to be associated, along with miR-34c, with aortic stiffening in human subjects. Conversely, miR-92a and miR-181b decrease in mouse aorta with aging and miR-92a is also decreased in human blood with aging.
Based on these initial observations, investigators examined whether mimicking these changes is sufficient to produce stiffening. Using a transfection-based approach in vivo, it was found that elevating miR-203 leads to arterial stiffening. Furthermore, administering an antagonist of miR-92a to mice increases PWV. To examine the effect of decreased miR-181b on aortic stiffness, researchers used mice carrying a deletion of the locus (miR-181a1/b1) that blocks expression of both miR-181a and miR-181b in aorta. These mice showed a premature onset of arterial stiffness as young adults. Thus, these findings indicate that aging-associated changes in expression of these candidate microRNAs play a causal role in eliciting arterial stiffness.
Although several studies have demonstrated that mimicking the alteration of a candidate microRNA is sufficient to elicit arterial stiffness, to our knowledge, miR-181b is the only candidate microRNA for which interventions have reduced arterial stiffness. Our strategy for normalizing miR-181b levels in aorta emerged from our observation that this microRNA is one of those elevated in a cell line that had been subjected to knockdown of the translin (TN)/trax (TX) microRNA-degrading enzyme, suggesting that it was targeted by this enzyme. We reasoned that if a decrease in miR-181b levels plays a key role in driving LAS, then TN knockout (KO) mice might be resistant to developing arterial stiffness. As C57BL6 mice display aging-associated arterial stiffening, they provide a suitable animal model of this disorder. However, since that would require waiting until the mice are close to 18 months old, we first tested this hypothesis in a paradigm that elicits increased aortic stiffness in just a few weeks. In this streamlined paradigm, mice are switched from regular water to high salt water (HSW; 4% w/v), and their PWV measured on a weekly basis. Remarkably, we found that TN KO mice are resistant to developing increased PWV induced by this paradigm. Furthermore, exposure to HSW decreases levels of miR-181b in WT mice, but not in TN KO mice, consistent with the view that TN deletion confers protection from increased stiffness by blocking degradation of miR-181b.
As inhibiting the activity of the TN/TX microRNA-degrading enzyme confers protection from the development of aging-associated arterial stiffening, inhibitors of this enzyme may have translational potential. However, since the observed protection occurred in mice with a constitutive inactivation of TN/TX, it will be important to check if inhibition of this enzyme after the initial onset of aging-associated arterial stiffening can prevent the progression or even reverse this process. In addition, as TN/TX inhibition may also elevate other microRNAs, it might be more advantageous to explore other strategies to increase miR-181b levels selectively. However, since extremely high elevations of miR-181b have been implicated in the development of atherosclerotic plaques, it may be important to identify strategies that reverse the age-associated decline in miR-181b levels without elevating them further into the range associated with pathological effects.