Author + information
- aSaha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, Kentucky
- bDepartment of Family Medicine, University of Kentucky College of Medicine, Lexington, Kentucky
- cDepartment of Surgery, University of Kentucky College of Medicine, Lexington, Kentucky
- dDepartment of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky
- ↵∗Reprint requests and correspondence:
Dr. Alan Daugherty, Saha Cardiovascular Research Center, University of Kentucky, B243 BBSRB, 138 Leader Avenue, Lexington, Kentucky 40536-0509.
Aortopathies have a wide range of causes. Ascending aortic aneurysms, for example, are linked to the presence of a bicuspid aortic valve (BAV), a common congenital abnormality (1). Although BAV-associated aortopathies frequently occur with devastating health consequences, there is a surprising paucity of information on disease mechanisms. Currently, clinicians rely on surgical approaches to reduce aortic rupture (2). Absence of validated medical therapies is mainly attributable to a lack of understanding of disease mechanisms. Therefore, any significant mechanistic findings may substantially influence development of new therapies.
Micro ribonucleic acids (miRNAs) are small noncoding RNAs that potentially regulate a diverse array of diseases. In the area of ascending aortic aneurysms, several recent studies have demonstrated changes in the expression of many different miRNAs (3–6). In this issue of the Journal, Wu et al. (7) have expanded the repertoire of miRNAs potentially involved in thoracic aortic diseases by demonstrating correlations of miRNA-17 (miR-17) with BAV-associated aneurysms. In this study, microarrays were performed using surgically excised ascending aortas in regions where dilation was defined as either “severe” or “lesser.” Several miRNAs were increased in the lesser region, with the authors electing to focus on miR-17 and related miRNAs.
Microarray data were validated using quantitative PCR in analyses that also included comparisons to tissues surgically excised from nonaneurysmal patients. miR-17 increases were restricted to adventitial and intimal regions of diseased segments. Regions of lesser dilation also had significant reductions in both the tissue inhibitors of metalloproteinase (TIMP)-1 and the TIMP-2 messenger RNA (mRNA) and protein. TIMP-3 protein, but not its mRNA, was reduced. Two of the proteases inhibited by TIMPs, matrix metalloproteinase (MMP)-2 and -9 were not changed at either the mRNA or protein level. However, there was a greater presence of proteolytic activated MMP-2. Collagen was more abundant in severe (than in lesser) aneurysmal regions, whereas abundance of elastin was the reverse. Although activated MMP-2 was greater in the lesser regions, elastase activity was lower, inferring that MMP2 abundance was not directly correlated to elastin integrity. A link between miR-17 and proteolysis was derived from studies that demonstrated TIMP-1 and TIMP-2 were regulated in smooth muscle cells (SMCs) by enhancing or inhibiting miR-17 activity. miR-17 regulated TIMPs in SMCs derived from severe, lesser, or nonaneurysmal regions.
Overall, these studies are consistent with findings that increased abundance of the miR-17 family and their regulated proteases contribute to BAV-associated ascending aortic disease.
As can be gleaned from this description, foundational data from the study by Wu et al. (7) were derived from a comparison of diseased tissue to regions with no or minimal pathology. Certainly, comparison of aneurysmal with nonaneurysmal tissue is a frequently used approach, but it has inherent shortcomings with no obvious resolution. One issue is the difficulty in acquiring meaningful control tissue, most commonly defined as nonaneurysmal aortic tissue from the same region as acquisition of diseased tissue. Acquisition of aortic buttons during coronary artery bypass enables collection of age- and sex-matched nonaneurysmal tissue from the proximal portion of ascending aorta. This acquisition approach permits comparison of tissues from the same region of the ascending aorta from different individuals; however, there are potential confounding variables, such as individual-specific conditions and comorbidities.
Wu et al. (7) tried to overcome these confounders, but they did not provide quantitative definitions of “severe” and “lesser.” Although this approach surmounts problems of comparisons across individuals, it relies on the premise that aneurysmal disease occurs by equivalent mechanisms throughout the ascending aorta. One potential confounder of this premise is heterogeneity of cell populations throughout the ascending aorta. The ascending aorta is a mosaic composed of SMCs derived from 2 different embryonic origins (second heart field and cardiac neural crest) (8–10), thereby leading to functional heterogeneity in this aortic region. It is worth noting that adventitia of the ascending aorta also displays heterogeneity along its length (10). In addition to cellular heterogeneity, mechanical stresses differ along the length of the ascending aorta (11). Therefore, further insight is needed to determine the relative merits of tissue acquisition from the same area from aneurysmal versus nonaneurysmal individuals, compared to acquisition of different aneurysmal locations in the same individual.
This study also determined the abundance of matrix proteases that are regulated during aortopathy beyond miR-17 and related miRNAs. A common pathological feature of thoracic aortic aneurysms is elastin degradation. This observation provides a logical rationale for focusing on MMP-2 and MMP-9, as both possess elastolytic activity. Indeed, these proteases have been the focus of studies of ascending aortic aneurysms (12). As unabated elastin degradation is undesirable, MMPs have a complex regulation system involving proteolysis, endogenous inhibitors, and removal mechanisms. TIMPs, endogenous inhibitors of MMPs, were decreased in the area of lesser dilation. The relative tissue levels of these proteins imply proteolytic regulation in the aortic tissue. However, the ability of TIMPs to inhibit MMPs requires their colocalization. Therefore, it is critical to determine whether the spatial distribution of MMPs and TIMPs is consistent with local regulation.
The ability of miR-17 to directly regulate TIMPs was demonstrated in primary SMCs cultured from aortic tissue. Although miR-17 manipulations regulated TIMP expression in these cells in vitro, primary increases in miR-17 were in the adventitia and intima of the lesser region in vivo. This location of miR-17 may be indicative of the complexity of ascending aortic aneurysms in which the primary manifestation of the medial pathology is influenced by other layers of the aorta. Certainly, there is an evolving recognition of a role for the adventitia in thoracic aortic disease (13). Although studies of cells isolated from ascending aortic tissue focused on SMCs, it will be helpful if future studies encompass a wider array of other vascular cell types.
Other reports have described numerous factors that differ in abundance between normal and aneurysmal tissue. Although data suggest some potential therapeutic targets, there should be a cautionary note in interpretations. By definition, this approach compares tissue of very different cellular compositions. Diseased tissues acquired at surgical intervention have highly advanced pathology. Therefore, it is not surprising that comparison of tissues at different disease states yields a wide spectrum of differences in cellular and protein contents. This provides valuable information on the presence or absence of biological entities. However, a comparison between diseased tissue and nondiseased (or less diseased) tissue does not discern whether changes are a consequence or cause. This shortcoming is magnified by the absence of tissues for analyses in the formative stages of disease, preventing comparisons before gross pathological changes have evolved. Surgical acquisition of tissue in the formative stages is an insurmountable barrier. Autopsy-based acquisitions also are logistically challenging. Hence, knowing whether biological entities are the cause or consequence of disease is the classically unanswered chicken-and-egg situation. Given this uncertainty, there should be some restraint in being overly zealous regarding mechanistic interpretation of changes in the abundance of miRNAs and their related proteins in a comparison between normal and diseased tissues.
Although human tissue is used to characterize the presence of different biological entities in aneurysmal versus nonaneurysmal tissue providing potentially useful initial directions to determine mechanisms, the challenge now is to provide insight into whether these miRNAs are directly involved in aneurysm formation. The most direct manner in which mechanistic information can be obtained is manipulation of miR-17 in an animal model of BAV-associated ascending aortic aneurysms. An animal model not only enables determination of efficacy of interventions but also permits tissue acquisition on a temporal basis to provide insight into the disease’s initiation and progression characteristics. As the authors note, there is no generally available animal model at the present time. Given the prevalence of BAV-dependent ascending aortic aneurysms and lack of validated medical therapy, such studies highlight the need to develop appropriate animal models relevant to human disease. Determining the importance of miRNAs in development of BAV-associated ascending aortic aneurysms will require a continuum of cell, animal, and human studies to define disease mechanisms.
↵∗ Editorials published in Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology.
The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
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