Specific circulating microRNA signature of bicuspid aortic valve disease

Bicuspid aortic valve (BAV) represents the most common congenital cardiac malformation and is commonly associated with the development of aortic valve dysfunction and the progressive dilation of the ascending aorta. In patients with BAV, the dilation of the aorta is associated with aortic regurgitation [1] and the risk of aortic dissection and thus often requires prophylactic aortic surgery [2‐6]. The causes of aorta dilation have been debated for several years, and changes in the flow characteristics within the ascending aorta have been suggested as the main cause [7, 8]. However, the mechanisms involved have not been fully elucidated.

MicroRNAs (miRNAs) are endogenously expressed 19–23nt-long noncoding RNAs (ncRNAs) that silence gene expression at the post-transcriptional level, largely via imperfect base-pairing interactions that occur preferentially within the 3′ untranslated regions (UTRs) of target mRNAs. Due to the ability of miRNAs to target hundreds of mRNAs, they are considered as potent post-transcriptional regulators, and in fact, it is suggested that a significant portion of the human genome is regulated by miRNAs. Aside from their intracellular function, miRNAs can be exported or released by cells into the circulating blood in highly stable forms [9]. miRNA signatures have been proposed as potential biomarkers with the potential to improve disease diagnosis and prognosis in clinical practice and have been identified as useful biomarkers for a wide range of cardiovascular diseases, including cardiovascular calcification [10], vulnerable coronary artery disease [11], heart failure [12], myocardial infarction [13], abdominal aortic aneurysm [14] and coronary artery disease [15]. Despite the large number of miRNAs identified thus far, the biological roles of most miRNAs and the molecular mechanisms underlying their function remain largely unknown.

In this study, we applied a miRNome-wide microarray approach using the plasma of healthy tricuspid aortic valve (TAV) individuals and BAV patients [with or without aortic dilation (BAV_dil)] to compare and identify specific miRNAs associated with BAV and the dilation of the ascending aorta. In a second stage, the expression levels of the miRNA candidates were validated in an independent cohort, providing new insights into the complex mechanisms that underlie BAV and aortic dilation.

This study found that the miRNA expression pattern in plasma is influenced by the morphology of the aortic valve and the dilation of the ascending aorta. Using comprehensive miRNA expression profiling and further RT-qPCR validation, we identified a circulating miRNA signature, including miR-122, miR-130a and miR-486, whose regulation is dependent on the tricuspid or bicuspid morphology of the aortic valve. Furthermore, the plasma expression of miR-718 may be a biomarker of aortic dilation, that is, an increased ascending aorta diameter is associated with the down-regulation of miR-718. We explored the biological implications of the differential expression of these miRNAs based on target gene prediction and the putative biological functions and processes regulated by the target genes. In this manner, the differential modulation of the bicuspid valve morphology-associated miRNAs might affect the TGF-β₁ signalling pathway, and the dysregulation of miR-718 might be associated with the progression of aortic dilation by modulating the focal adhesion and blood vessel remodelling processes.

The molecular features and mechanisms that underlie BAV and the dilation of the aorta are poorly understood, and to our knowledge, the miRNA expression profile and putative gene regulation in BAV disease and aortic dilation identified in this study by screening in plasma of BAV patients has not been previously reported. Previous studies have analysed the expression of miRNAs in aortic tissue segments of patients with BAV and animal models. Nigam et al. [28] determined that miR-26a, miR-30b and miR-195 were decreased in the aortic valve leaflets of patients requiring aortic valve replacement due to aortic stenosis compared to those requiring replacement due to aortic insufficiency, and both pathologies are associated with BAV. Using porcine valvular interstitial cells and human valve leaflets, Yanagawa et al. [29] determined that the down-regulation of miR-141 in stenotic bicuspid leaflets regulates BMP-2-mediated valve calcification. Wu et al. [30] performed a paired comparison of the miRNA expression between severely dilated and normal-appearing, less-dilated aortic samples, associating the differential regulation of the miR-17 gene cluster with the predisposition to dilation through the dysregulation of the matrix metalloproteinases-tissue inhibitors of matrix metalloproteinases (TIMP-MMP) pathway. However, although the determination of the miRNA expression profile in tissue samples provides valuable information regarding the pathophysiological mechanisms underlying diseases, this expression signature often cannot be extrapolated to the bloodstream. Thus, unlike these tissue-specific miRNAs, the determination of a circulating miRNA expression profile allows for the integration of the complexity associated with multiple tissues, as is in the case of complex diseases, in addition to serving as biomarkers of these diseases.

In clinical practice, the diagnosis of BAV and ascending aorta dilation is currently performed exclusively based on imaging techniques (i.e., echocardiography, TAC, and magnetic resonance) and the subsequent interpretation by an expert. Moreover, both BAV and the dilation of the aorta are normally presented as asymptomatic clinical conditions, and the molecular causes underlying both of these pathologies are unknown. Therefore, determining plasma molecular biomarkers of BAV and the progression of aortic dilation would be extremely useful in expanding the basic knowledge of the aortic dysfunction, as well as for the identification of new therapeutic targets that might significantly improve the diagnosis, prognosis and treatment of BAV and aortic dilation. Moreover, the determination of biomarkers in the plasma has an added value because in the plasma, the miRNA levels are reproducible and consistent among individuals, and the miRNAs appear to be protected from endogenous ribonuclease-induced degradation [31]. In addition, in contrast to other tissues, the plasma is more easily accessible by non-invasive means, which might improve the feasibility of these molecules as useful biomarkers in routine clinical practice.

The bicuspid morphology of the aortic valve is associated with an abnormal blood flow and shear stress at the ascending aorta that leads to damage and apoptosis of endothelial cells at the cellular level, involving the activation of multiple biological processes, such as endothelial dysfunction, inflammation, oxidative stress, angiogenesis and extracellular matrix remodelling [32‐34]. Considering this BAV-associated environment, we hypothesized that circulating miRNAs might be involved in the paracrine communication between cells and contribute to the regulation of disease progression, and thus, the circulating levels of miRNAs might reflect the hemodynamic alteration of the blood flow resulting from the bicuspid morphology of the aortic valve.

In addition to their role as biomarkers for various diseases, miRNAs can be considered potential dynamic master regulators of signalling pathways due to their capacity to specifically control the expression of key components of signal transduction at multiple levels. Therefore, based on a functional analysis, our data supported the crucial role of the dysregulation of genes involved in the TGF-β₁ signalling pathway in BAV disease [35]. In addition, we propose that miR-122, miR-130a and miR-486 might serve as molecular effectors in the BAV associated dysregulation of TGF-β₁ receptor. TGF-β₁ is a crucial factor mediating tissue fibrosis and extracellular matrix (ECM) remodelling in ascending aorta. TGF-β₁ is a pleiotropic cytokine secreted and stored in the ECM as a biologically inactive form that is complexed with latent TGF-β₁ binding protein (LTBP-1) in the ECM. Activated TGF-β₁ binds to a heterodimeric receptor complex comprising two subunits (TGFβR1 and TGFβR2) whose genes are targeted by miR-122 and miR-130a, respectively, activating Smad proteins, which mediates the intracellular signalling of TGF-β₁ [36]. The phosphorylation of SMAD2 (the gene targeted by miR-122) and SMAD3 results in the formation of heterooligomeric complexes with SMAD4 (the gene targeted by miR-122, miR-130a and miR-486). TGF-β₁ signalling has been reported to play divergent roles in aortic dilation depending on its location (i.e. abdominal or thoracic aortic aneurysms) or aortic valve morphology [37]. As limitation of this study, the role of these miRNAs in the regulation TGF-β1 signalling pathway was determined based on computational predictions and it was not experimentally confirmed.

We can speculate that the opposite regulation of mir-122 (decreased expression levels) and miR-130a (increased expression levels) that we observed could be associated with the imbalance of TGFβR1 and TGFβR2 subunits. This speculation is supported by previous findings where the postnatal disruption of the Tgfbr2 gene expression was associated with decreased canonical TGF-β₁ signalling and accelerated aneurysm growth in a murine model of Marfan syndrome [38]. Moreover, Forte et al. [39], reported that ascending aorta aneurysms in BAV patients was associated with an imbalance between the TGFβR1 and TGFβR2 subunits, although, in that case the authors associated this imbalance by an intrinsic defect of TGFβR1 expression. This imbalance between both subunits of the TGF-β₁ receptor could be indicating a redirection towards the activation of the non-canonical TGF-β₁ signalling [37], which is related to the induction of the ECM degradation and MMP activity, both biological processes associated with BAV and aortic dilation.

In addition to the biological and cardiology-relevant implication of each miRNA individually, miR-122 accounts for 70% of the total miRNAs in the adult liver [40] and is considered a central player in liver biology and in the regulation of cholesterol and fatty acid metabolism [41]. Interestingly, Beaumont et al. [42] demonstrated that the down-regulation of miR-122 was associated with the up-regulation of TGF-β₁ expression, increasing the severity of myocardial fibrosis in patients with aortic stenosis, which is the most frequent valvular complication in BAV patients [43]. miR-130a has important roles in the cardiovascular system due to its capacity to promote the proliferation of endothelial cells and vascular smooth muscular cells. In fact, miR-130a has been described as a regulator of the angiogenic phenotype of vascular endothelial cells through its ability to modulate the expression of the homebox genes GAX and HOXA5 [44]. Furthermore, the expression of miR-130a in the aorta is correlated with vascular remodelling in spontaneously hypertensive rats (SHRs) [45]. Regarding miR-486, Holliday et al. [46] demonstrated that the expression of miR-486 in human aortic valvular endothelial cells was shear dependent, and Navon et al. [47] suggested that a reduction of miR-486 supports tissue remodelling characterized by increased proliferation. Therefore, our findings regarding the miRNA signature associated with the morphology of the aortic valve support the notion that miR-122, miR-130a and miR-486 play a key role in BAV disease and add new knowledge regarding the molecular effectors of the TGF-β ₁ pathway dysregulation associated with BAV.

We propose miR-718 as a new non-invasive biomarker of dilation of the ascending aorta. We demonstrated its potential as molecular feature associated with an aortic dilation in patients with a dilated ascending aorta that was independent of the tricuspid or bicuspid morphology of the aortic valve. We found only 4 references [48‐51] in PubMed when searching in the literature for information on the biological function of miR-718; to date, no one has referred to the biological function of miR-718 expression in the cardiovascular field, including its function in relation to aortic valve morphology or the dilation of the aorta.

Using a bioinformatics approach, we predicted the putative target genes of miR-718 and the biological function and processes that it might regulate. The miR-718 target genes were significantly enriched for the “blood vessel remodelling” process and “focal adhesion pathway”. Ascending aorta dilation is defined as a degenerative vascular disorder due to the destructive remodelling of the aortic wall and the degradation of the ECM proteins, leading to the recruitment and infiltration of immune system cells mediated by the secretion of adhesion molecules. Interestingly, vascular endothelial growth factor A (VEGFA) is one of the miR-718 target genes that was significantly enriched for the “focal adhesion pathway”. VEGFA acts on endothelial cells by increasing vascular permeability and angiogenesis, inducing vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis [52]. These biological implications of the miR-718 target genes might explain the relation between miR-718 and ascending aorta dilation.

In BAV, as in other disease-associated complications, the emergence of the ascending aorta dilation cannot be considered as a discrete alteration in time; instead, it is a continuous process associated with the pathophysiological implications of BAV, and such a complex process involves multiple cell signalling pathways that might be integrated to reflect the complexity of the disease. In this manner, our results are consistent with previous findings that postulated the crosstalk between TGF-β₁ and VEGF together with the Notch signalling pathways, to act coordinately in space and time in the regulation of vascular morphogenesis [53]. With this consideration, our circulating miRNA signature, including miR-122, miR-130a, miR-486 and miR-718 expression, integrates the complexity associated with not only the bicuspid morphology of the aortic valve but also the progressive dilation of the ascending aorta. Furthermore, whether these miRNAs are directly involved in the aortic wall pathogenesis of BAV disease or if they are a consequence of the increased aortic shear stress generated by the anomalous aortic flow caused by BAV remains unknown.

We proposed miR-122, miR-130a, miR-486 and miR-718 as new molecular features associated with BAV and aortic dilation by the regulation of TGF-β₁ and vascular remodelling mediated by the VEGF signalling pathway.