MicroRNA-125b and chemokine CCL4 expression are associated with calcific aortic valve disease.

Abstract Calcific aortic valve disease (CAVD) is a progressive pathological condition with no effective pharmacological therapy. To identify novel molecular pathways as potential targets for pharmacotherapy, we studied microRNA (miRNA) profiles of heavily stenotic aortic valves (AS). One of the most upregulated miRNAs in AS valves compared to control valves was miR-125b (1.4-fold; P < 0.05). To identify CAVD-related changes in gene expression, DNA microarray analysis was performed, including an intermediate fibro(sclero)tic stage of the disease. This revealed changes especially in genes related to inflammation and immune response, including chemokine (C-C motif) ligand 3 (CCL3) and 4 (CCL4). CCL3 mRNA level was increased 3.9-fold (P < 0.05) when AS valves were compared to control valves, and a 2.5-fold increase (P < 0.05) in CCL4 gene expression was observed when fibro(sclero)tic valves were compared to control valves. Both CCL3 and CCL4 localized to macrophages by immunofluorescence. To identify chemokine–miRNA target pairs, data from miRNA target prediction databases were combined with valvular miRNA and mRNA expression profiles. MiR-125b was computationally predicted to target CCL4, as confirmed experimentally in cultured human THP-1 macrophages. Collectively, miR-125b and CCL4 appear to be involved in the progression of CAVD and may offer novel therapeutic and diagnostic strategies related to this disease.


Introduction
Calcifi c aortic valve disease (CAVD) is the most common form of valvular heart disease in the Western world. Milder degrees of aortic valve calcifi cation, i.e. aortic sclerosis, can be seen in 26% of people aged Ͼ 65 years, whereas severe calcifi cation with impaired valve leafl et motion (termed aortic stenosis, AS) has a prevalence of 2% (1). CAVD leads to a signifi cantly increased risk of mortality; currently the only treatment for symptomatic CAVD is surgical or transcatheter aortic valve replacement (2). Given that the majority of patients are elderly and currently the only eff ective treatment to CAVD is valve replacement, there is an increasing demand for a pharmacologic intervention.
Calcifi cation of the aortic valve was formerly considered to be an age-related degenerative process, but emerging evidence has revealed it as an actively regulated disease (3) and the outcome of several overlapping processes, including calcifi cation, ossifi cation, neovascularization, and infl ammation (3). Th ese processes are also typical determinants of atherosclerosis, but despite pathophysiological similarities only about 50% of patients with CAVD have clinically signifi cant atherosclerosis (4). Although some progress has been made in understanding the factors driving CAVD, the molecular pathogenesis and signaling mechanisms underlying the progression of AS are poorly understood.
MicroRNAs are short, non-coding RNA molecules that generally function as negative regulators of gene expression by complementary binding of 3 ' -UTRs of messenger RNAs (mRNAs). Th is can lead to either blockade of protein translation or the complete degradation of the miRNA -mRNA complex. It has been estimated that miRNAs may regulate up to one-third of the human genome (5). Th e involvement of miRNAs in cardiovascular diseases ranges from primary risk factors to pathophysiological processes (6). However, the involvement of miRNAs in the pathogenesis of AS remains to be uncovered.
Calcifi c aortic valve disease (CAVD) is a progressive pathological condition with no eff ective pharmacological therapy. To identify novel molecular pathways as potential targets for pharmacotherapy, we studied microRNA (miRNA) profi les of heavily stenotic aortic valves (AS). One of the most upregulated miRNAs in AS valves compared to control valves was miR-125b (1.4-fold; P Ͻ 0.05). To identify CAVD-related changes in gene expression, DNA microarray analysis was performed, including an intermediate fi bro(sclero)tic stage of the disease. This revealed changes especially in genes related to infl ammation and immune response, including chemokine (C-C motif) ligand 3 (CCL3) and 4 (CCL4). CCL3 mRNA level was increased 3.9-fold ( P Ͻ 0.05) when AS valves were compared to control valves, and a 2.5-fold increase ( P Ͻ 0.05) in CCL4 gene expression was observed when fi bro(sclero) tic valves were compared to control valves. Both CCL3 and CCL4 localized to macrophages by immunofl uorescence. To identify chemokine -miRNA target pairs, data from miRNA target prediction databases were combined with valvular miRNA and mRNA expression profi les. MiR-125b was computationally predicted to target CCL4, as confi rmed experimentally in cultured human THP-1 macrophages. Collectively, miR-125b and CCL4 appear to be involved in the progression of CAVD and may off er novel therapeutic and diagnostic strategies related to this disease.
In this study we compared miRNA profi les of stenotic and control aortic valves to determine the miRNAs involved in the development of CAVD. To identify the possible miRNA targets, a genome-wide gene expression analysis of aortic valves was performed. Th is DNA microarray analysis revealed changes in valvular gene expression associated especially with infl ammation, chemokines and their receptors being the dominant group of genes upregulated in calcifi c aortic valves. Next, to identify miR-NA target pairs with confi dence, we combined data from miRNA target prediction databases and expression profi les of miRNA and mRNA focusing on infl ammatory response. We found that miR-125b was upregulated in stenotic valves, and it was found to target chemokine (C-C motif) ligand 4 (CCL4) in silico ; this relationship was further confi rmed in vitro in cultured human THP-1 macrophages.

Materials and methods
An expanded Methods section is available in the supplementary material online: patient demographics (Supplementary Table 1, to be found online at http://informahealthcare.com/doi/abs/10. 3109/07853890.2015.1059955), analysis of miRNA, miRNA array, DNA microarray, pathway analysis, analysis of RNA, Bioinformatic microRNA target prediction, miRNA transfection, ELISA, histological and immunohistological analysis, and statistical analysis.

Results
Valvular calcifi cation and neovascularization are markedly elevated in patients with aortic stenosis.
All the valves in the AS group were markedly calcifi ed, the proportion of calcifi ed area to the total aortic valve area being 39.6% Ϯ 3.1% ( Figure 1A), while only weak calcifi cation was observed in the fi bro(sclero)sis group. Th ere were no macroscopic signs of calcifi cation in the control group. Factor VIII immunostaining was used to visualize the density of neovascularization in aortic valves. Neo-angiogenesis was signifi cantly activated in the AS group in comparison to other groups, while only few vascular structures were found in control and fi bro(sclero)tic valves ( Figure 1B).

DNA microarray analysis of micro-RNA targets genes in CAVD
To identify potential miRNA target genes, we determined diff erentially expressed genes during aortic valve calcifi cation by evaluating gene expression profi les of control, fi bro(sclero)tic, and stenotic valves ( n ϭ 5 in each group). Selected DNA microarray results were confi rmed by comparison with mRNA levels obtained by quantitative RT-PCR; both methods showed changes of equal magnitude (Supplementary Table 4, to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2015.1059955).
Our analysis identifi ed 302 up-and 248 downregulated transcripts (fold-change Ն 2-fold) when the AS group was compared to the control group ( Figure 3A; and Supplementary Table 5, to be found online at http://informahealthcare.com/ doi/abs/10.3109/07853890.2015.1059955). Th e majority of upregulated genes were genes related to infl ammation and immune response, as well as to signal transduction. In contrast, the majority of downregulated genes with known function were signal transduction proteins and regulators of transcription ( Figure 3B). Th e top 10 most up-and downregulated genes in stenotic versus control valves are shown in Table I. Several chemokines were among the top upregulated genes during development of AS ( Figure 3B). Importantly, the majority of changes in gene expression was seen at the late phase of the disease: 235 genes were upregulated and 170 genes downregulated more than 2-fold when the AS group was compared with the fi bro(sclero)tic group ( Figure    Localization of CCL3 and CCL4 in aortic valve cusps was evaluated by immunofl uorescence revealing that CCL4 is expressed in macrophages ( Figure 5A). CCL3 is expressed in some macrophages as well as myofi broblasts ( Figure 5B and C). Th ese results were verifi ed using immunohistochemistry (Supplementary Figure 5, to be found online at http://informahealthcare. com/doi/abs/10.3109/07853890.2015.1059955).

Discussion
CAVD is recognized as a complex, actively regulated biological process including infl ammation, lipid retention, and calcifi cation (3). Th erefore, a better understanding of the molecular mechanisms driving CAVD pathogenesis in aortic valves is needed to provide new targets for novel therapeutic interventions. In this study we performed miRNA profi ling of normal and calcifi ed aortic valves to determine involvement of miRNAs in the pathogenesis of CAVD. We characterized targets of these miRNAs by conducting genome-wide gene expression profi ling of diff erent stages of CAVD. Th e most prominent changes were observed in genes involved in infl ammatory and immune response including chemokines CCL3 and CCL4. We identifi ed the miRNAs regulating these infl ammatory genes by combining data from miRNA target prediction databases and expression profi les of both miR-NA and mRNA. We found that miR-125b targeted CCL4, and a miR-125b mimic decreased expression of CCL4 also in THP-1 macrophages. Altogether, our results suggest that miR-125b and CCL4 are associated with the progression of CAVD.
As a major fi nding, we detected a signifi cant downregulation of miRNAs -939, -602, and -374b * as well as increased expression of miR-125b in calcifi ed aortic valves. In further analysis we focused on miR-125b, previously linked to infl ammation (7) and calcifi cation (8), the two key processes of CAVD. Other miR-NAs identifi ed by our array may also have important regulatory potential. For example, miR-939 was recently shown to regulate nitric oxide synthase, an important factor in endothelial function (9). MiR-374b may be a regulator of CCTTA enhancer binding protein-β (C/EBP-β ) (10), a key factor in macrophage polarization (11). Also, miR-602 could be a regulator of Ras association domain-containing protein 1 (RASSF1) (12), which has been (to be found online at http://informahealthcare.com/doi/abs/ 10.3109/07853890.2015.1059955).
Th e complete list of signifi cantly up-or downregulated genes in stenotic versus control valves was loaded into the STRING database to identify potential interactions between diff erentially expressed genes. Of those genes, 103 had confi rmed regulatory connections with biological evidence in databases (list of genes in Supplementary Table 8

MiRNA-125b modulates chemokine CCL4
To identify miRNA target pairs with confi dence, we combined data from miRNA target prediction databases and expression profi les of both miRNA and mRNA with focus on infl ammatory response. DNA microarray analysis showed that especially many chemokines such as CCL3 and CCL4 were signifi cantly upregulated in CAVD. Interestingly, one of the most prominent dysregulated miRNAs was miR-125b, which has previously been linked to regulation of infl ammatory genes in vascular smooth muscle cells (VSMCs) of diabetic mice (7).
Quantitative RT-PCR analysis confi rmed that CCL3 and CCL4 mRNA levels were signifi cantly increased in the diff erent stages of CAVD ( Figure 4A and B). According to MicroCosm targets, CCL4 may be regulated by miR-125b ( Figure 4C). To test this hypothesis, we transfected human THP-1 macrophages with a mimic for miR-125b and measured the corresponding mRNA and protein levels of CCL4. As shown in Figure 4D, a statistically signifi cant decrease in CCL4 protein secretion was seen in response to miRNA-125b transfection as measured by ELISA, which corresponded to changes in the CCL4 mRNA levels (Supplementary  further shown to regulate apoptosis (13). Th ese fi ndings suggest that miR-dysregulation during CAVD may have multifactorial impact on the underlying pathological processes. In search of possible miRNA targets, our genome-wide gene expression analysis demonstrated that the majority of upregulated genes were involved in activation of infl ammatory and immune response in each phase of the disease process, chemokines and immunoglobulins being the largest functional families. Th is supports previous studies emphasizing the importance of chronic infl ammation in AS. For example, infl ammatory infi ltrates within stenotic valve leafl ets have been associated with an active remodeling process (14). Our pathway analysis further highlighted the biological relevance of infl ammatory response in CAVD since chemokines and their receptors as well as mediators of the Tcell response such as CD247 were central signaling molecules in this network. To our knowledge, there is only one earlier largescale gene expression study of CAVD (15). Intriguingly, matrix metallopeptidase 12, CXCL5, tryptophan 2,3-dioxygenase, and collagen XI α 1 were among the top 10 upregulated genes in both the earlier and our study. We also identifi ed other well-known regulators of CAVD including cathepsin S (16), tryptase (17), as well as osteopontin, bone sialoprotein II, and osteoprotegerin (18), in agreement with the established role of extracellular matrix modulation in the pathophysiology of CAVD (15). Of note, in the study of Bosse et al. (15) only heavily stenotic and control valves were compared, while in our study an intermediate fi bro(sclero) tic group enabled studying earlier stages of CAVD. Th erefore, the important fi nding of our study is that there were only minor changes at the gene expression level in the early phase of the disease. Th is implies that the irreversible pathological changes occur in the later stages of the disease progression, underscoring the importance of early intervention and prevention of CAVD.
Most of the genes we uncovered have not previously been known to be associated with CAVD, although they are linked to the key processes of atherosclerosis, e.g. angiopoietin-1 to angiogenesis (19) and BCL2-related protein A1 to apoptosis (20). In addition, we identifi ed several transcription factors, including runt-related transcription factor 3 (Runx3), activation transcription factor-3 (ATF-3), and interferon regulatory factor 8 (IRF-8), which could be of importance for the development of CAVD. Runx3 is an osteogenic gene regulating e.g. expression of osteopontin (21), ATF-3 has been linked to macrophage activation (22), and IRF-8 is a transcription factor that aff ects infl ammatory cell populations in atherosclerosis (23). Clearly, further studies are needed to clarify their role in pathogenesis of CAVD.
In this study we detected an increased expression of the infl ammatory chemokines CCL3 and CCL4. Although the expression of several chemokines such as CCL18 (24) and CCL23 (25) has been linked to the development of atherosclerotic lesions in humans, the role of chemokines in CAVD is largely unknown and limited to expression studies (15,26). In addition, there is evidence that CCL4 could also function as a heterodimer with CCL3 (27), which was also upregulated in stenotic valves. Similarly, the expression of ATF-3 and CCR5 was upregulated in our array. ATF-3 has been shown to regulate CCL4 expression (28), and CCR5 is also a receptor for CCL4 (29). Our immunofl uorescence studies of CCL3 and CCL4 support the evidence that these chemokines localize in macrophage-rich microenvironments, possibly being secreted by the macrophages in order to attract more infl ammatory cells into the infl amed valve (30). We also demonstrate

Study limitations
In addition to the chemokine regulation presented here, the eff ects of miRNAs in aortic valve surely extend to hundreds of genes. Mapping these interactions further will provide a more comprehensive understanding of CAVD progression. To our knowledge, the only previous study on miRNAs in aortic valve leafl ets was limited to a small population of congenitally bicuspid aortic valves, and the authors were unable to detect any genes targeted by the identifi ed miRNAs (35). It should also be noted that there was some heterogeneity between patient groups, including age. Nevertheless, for both miRNA and mRNA arrays, the valves were matched for age, sex, and treatments. Finally, the miRNAtargeted transcription was studied only in THP-1 macrophages. Based on our immunofl uorescence-based localization studies, macrophages were the likely source of CCL4, although we cannot rule out the contribution of other cell types at tissue level.

Conclusions
In human stenotic aortic valves miR-125b is upregulated, whereas miR-939, miR-602, and miR-374b * are downregulated, revealing that miRNAs are associated with the progression of CAVD. We showed that chemokines are among the most upregulated infl ammatory genes in CAVD, and experimental evidence demonstrated that miR-125b is one of the regulators of CCL4 in THP-1 macrophages. Altogether, our data suggest a novel miRNA-mediated that CCL3 is in part expressed by myofi broblasts as a part of the infl ammatory response. Interestingly, it has recently been shown that modulation of the chemokine system may be an eff ective way to reduce atherosclerosis and infl ammation (31 -33), suggesting that pharmacological modulation of the chemokine system could be an eff ective treatment strategy in the development of CAVD. We discovered that CCL4 could be a potential target for miR-125b regulation, since the transfection of miR-125b into THP-1 macrophages signifi cantly reduced both CCL4 mRNA and protein levels. Interestingly, we discovered a concomitant increase of expression of both miR-125b and CCL4 in the valves, which could be due to the overall infl ammatory condition in the valvular tissue. In VSMCs, miR-125b has been shown to regulate infl ammatory genes (7) as well as vascular calcifi cation (8). Th erefore, it is likely that the tissue-level increase of miR-125b is related to the infl ammatory and osteogenic response of valvular cells, although it is a negative regulator of CCL4 in macrophages. Consequently, the upregulation of miR-125b may occur in cells other than macrophages. It has also been shown that under certain circumstances miRNAs may also induce the upregulation of target genes (34). Th is would provide an alternative explanation for the simultaneous upregulation of both miR-125b and CCL4. It is also very likely that CCL4 is regulated by a network of several factors which remain beyond the scope of this study. Further research is needed to determine the mechanism of this miRNA-mediated regulation in CAVD. Figure 5. Chemokine localization in aortic valves. A: CCL4 is expressed in macrophages. In this area, there is a strand of subendothelial macrophages as well as some scattered macrophages (arrows), in which the co-localization of CCL4 and CD68 is seen. Detailed image of a single macrophage is shown in the inset. B: CCL3 is expressed in some macrophages (arrow) and CD68-negative spindle cells (double arrows). Detailed image of a single macrophage is shown in the inset. C: In this focus, there are several α -SMA-positive myofi broblasts. Some of these co-express CCL3 (arrows). regulatory mechanism in CAVD and may off er novel therapeutic and diagnostic strategies related to this disease.