**3. miRNAs and cardiovascular disease**

Cardiovascular calcification is an independent risk factor for cardiovascular morbidity and mortality. Several risk factors can accelerate atherosclerosis and cardiovascular calcification, including age, hypercholesterolemia, metabolic syndrome, CRD, and T2D. Cardiovascular calcification can be distinguished by location — as intimal (atherosclerotic) , medial (CRD, T2D), or valvular [3]. Atherosclerotic calcification occurs as a part of atherogenic progress in the vessel intima. Small hydroxyapatite mineral crystals (microcalcification) can be visualized in early lesions [13]. Medial calcification occurs primarily in association with CRD and T2D, independently of hypercholesterolemia. Aortic valve calcification leads to impaired move‐ ment of aortic valve leaflets, and causes valve dysfunction [2]. All three processes shared risk factors and etiological factors, including inflammation and oxidative stress.

The identification of circulating miRNA as a novel biomarker in various diseases is a growing area of research investigation. Many pioneering studies describe specific miRNA patterns in

**Figure 1.** Schematic overview of miRNA biogenesis.

**2. Micro-RNA biology: Biosynthesis and function**

exons or within intergenic regions [7].

124 Calcific Aortic Valve Disease

**3. miRNAs and cardiovascular disease**

Micro-RNAs (miRNAs) are a large class of evolutionarily conserved, small, endogenous, noncoding RNAs serving as essential post-transcriptional modulators of gene expression [5]. miRNAs regulate biological processes by binding to mRNA 3'-untranslated region (UTR) sequences to attenuate protein synthesis or messenger RNA (mRNA) stability [6]. Acting as genetic switches or fine-tuners, miRNAs are key regulators of diverse biological and patho‐ logical processes, including development, organogenesis, apoptosis, and cell proliferation and differentiation. miRNA dysregulation often results in impaired cellular function and disease progression. It has been estimated that the whole human genome encodes for about 1000 miRNAs which may be located within introns of coding or non-coding genes, within host

miRNA biogenesis is shown in Figure 1. The transcription process is mediated by the RNApolymerase II that produces long precursor RNAs known as "primary miRNA" (pri-miRNA) with a typical hairpin morphology [8]. A nuclear endonuclease, called DROSHA, then crops the distal stem portion of pri-RNA obtaining shorter chains (pre-miRNA) [9]. Pre-miRNA is transported to the cytoplasm by the nuclear receptor Exportin-5 [10] and processed by DICER, an RNase III, to short double-stranded RNA sequence containing the miRNA and the 'star strand' (miRNA\*). miRNA\* is degraded after stripping the miRNA strand to obtain mature miRNA [11]. Mature miRNA interact with proteins like Argonaute endonuclease (Arg 2), in order to form the RNA-induced silencing complex (RICS), which directs mature miRNA

A single miRNA may modulate hundreds of miRNAs, and one mRNA has multiple predicted binding sites for miRNAs in their 3'UTR. Furthermore, after cleavage of a target mRNA, miRNAs are not Destroyed; so they may recognise and modulate other mRNAs [5, 12].

Cardiovascular calcification is an independent risk factor for cardiovascular morbidity and mortality. Several risk factors can accelerate atherosclerosis and cardiovascular calcification, including age, hypercholesterolemia, metabolic syndrome, CRD, and T2D. Cardiovascular calcification can be distinguished by location — as intimal (atherosclerotic) , medial (CRD, T2D), or valvular [3]. Atherosclerotic calcification occurs as a part of atherogenic progress in the vessel intima. Small hydroxyapatite mineral crystals (microcalcification) can be visualized in early lesions [13]. Medial calcification occurs primarily in association with CRD and T2D, independently of hypercholesterolemia. Aortic valve calcification leads to impaired move‐ ment of aortic valve leaflets, and causes valve dysfunction [2]. All three processes shared risk

The identification of circulating miRNA as a novel biomarker in various diseases is a growing area of research investigation. Many pioneering studies describe specific miRNA patterns in

towards the targeted mRNA and bind on their 3' untranslated region (UTR) [6].

factors and etiological factors, including inflammation and oxidative stress.

cardiovascular diseases. The first study reporting circulating miRNAs in patients with atherosclerosis was published in 2010, demonstrating a reduction of circulating vascular- and inflammation-associated miRNAs (miR-126, miR-17, miR-92a, miR-155) in patients with coronary artery disease (CAD) [14]. In addition, tissue levels of miRNAs were investigated.

Here we summarize and discuss the current knowledge on circulating and tissue miRNAs in diseases associated with cardiovascular calcification (Tables 1 and 2).

#### **3.1. miRNAs in coronary artery disease**

Studies about miRNA expression in calcified vessels are rare. Li *et al.* analyzed the expression of miRNAs in patients with peripheral artery disease (arteriosclerosis obliter‐ ans), characterized by fibrosis of the tunica intima and calcification of the tunica media [15]. miR-21, miR-130a, miR-27b, let-7f, and miR-210 were significantly increased, while miR-221 and miR-222 were decreased in the sclerotic intima, compared to normal vessels [15]. Higher levels of miR-21 and miR-210 were confirmed in a study that compared atherosclerotic with non-atherosclerotic left internal thoracic arteries [16]. In addition, the expression of miR-34a, miR-146a, miR146b-5p, and miR-210 increased more than 4-fold in atherosclerotic arteries. Several predicted targets were downregulated [16]. Another study found a different miRNA pattern using plaque material from the carotid artery, com‐ pared with the arteria mammaria interna as control tissue [17]. The healthy vessel ex‐ pressed higher levels of miR-520b and miR-105, whereas miR-10b, miR-218, miR-30e, miR-26b, and miR-125a were predominantly expressed in atherosclerotic plaque [17]. The investigators in both studies, however, did not examine miRNAs in calcified lesions. Microcalcification is thought to cause plaque rupture [18, 19]. Destabilized human pla‐ ques are characterized by a specific miRNA expression profile (high levels of miR-100, miR-127, miR-145, miR-133a, miR-133b). Target genes of these miRNAs (Nox1, MMP9, CD40) may play a role in vascular calcification [7]. Thus, miRNAs could participate in the formation of hydroxyapatite crystals, and thereby have an important role in regulating atherosclerotic plaque toward unstable phenotypes and rupture [20].

Fichtlscherer *et al.* authored the first study investigating circulating miRNA in CAD [14]. Plasma levels of miR-17, miR-92a, miR-126, miR-145, and miR-155 were reduced in CAD compared to healthy controls, whereas miR-133a and miR-208a were increased [14]. Another study demonstrated a positive correlation of plasma miR-122 and miR-370 levels with the presence and severity of CAD [21]. Both miRNAs were significantly increased in hyperlipi‐ demia patients, compared to controls [21]. Increased levels of miR-27b, miR-130a, and miR-210 were observed in the serum of arteriosclerosis obliterans patients [15].

Comparison of published studies is challenging mainly because of the different sources of circulating miRNAs, which include serum, whole blood, PBMCs, EPCs, and platelets (Table 1). The miRNA profiles obtained from the different studies, therefore, are often not the same. In this context, a recent report suggested the necessity of careful selection for reference miRNAs by showing that hemolysis may significantly affect the levels of plasma miRNAs previously used as controls [22].

Polymorphisms in the 3'UTR may alter miRNA binding, leading to post-transcriptional dysregulation of the target gene and aberrant protein level. Functional single-nucleotide polymorphisms (SNPs) of miRNA-binding sites associate with the risk of cardiovascular disease. Wu *et al.* discovered a SNP in the miR-149 binding site of the 5,10-methylenetetrahy‐ drofolate reductase (MTHFR) gene that associated with increased risk for CAD [23]. Further‐ more, a larger study in a Chinese population of 956 CAD patients and 620 controls revealed that a SNP in the binding sites for miR-196a2 and miR-499 associated with the occurrence and prognosis of CAD [24].

#### **3.2. miRNAs in diabetes and chronic renal disease (CRD)**

T2D is a major risk factor for cardiovascular disease. Zampetaki *et al*. identified a plasma miRNA signature for T2D that includes reduced levels of miR-223, miR-15, miR-20b, miR-21, miR-24, miR-29b, miR-126, miR-150, miR-191, miR-197, miR-320, and miR-486, and elevated levels of miR-28-3p [33]. Reduced miR-126 levels antedated diabetes manifestation, and might explain the impaired peripheral angiogenic signaling in patients with T2D. Reduction of circulating miR-21 and miR-126 was confirmed by Meng *et al.*, who also found a decrease of miR-27a,b and miR-130a in T2D patients [35]. Another study demonstrated mostly elevated miRNA levels (miR-9, miR-29a, miR-30d, miR-34a, miR-124a, miR-146a, and miR-375) in serum from T2D patients, compared with pre-diabetic and/or normal glucose tolerance

conditions [36]. In contrast, reduced miR-146a levels in PMBCs from Asian Indian T2D patients associated with insulin resistance, poor glycemic control, and several proinflammatory cytokine genes [34]. miR-146a participates in the transcriptional circuitry regulating fibronec‐

CRD, chronic renal disease; T2D, type 2 diabetes; CAD, coronary artery disease; AS, aortic stenosis; HC, familial hyper‐ cholesterolemia; AO, arteriosclerosis obliterans; PBMC, peripheral blood mononuclear cell; EPC, endothelial progenitor

**miRNA Disease Source Finding**

CAD Serum

T2D Plasma

HC HDL

miR-146a T2D PBMC Decreased [34]

miR-21, -27a, b, -126, -130a T2D EPC Decreased [35]

miR-16, -21, -155, -210, -638 CRD Plasma Decreased [37]

miR-21, -27b, -130a, -210 AO Serum Increased [15]

miR-9, -29a, -30d, -34a, -124a, -146a, -375 T2D Serum Increased [36]

miR-146a/b CAD PBMC Increased [25] miR-34a CAD EPC Increased [26] miR-221, -222 CAD EPC Increased [27] miR-135a, -147 CAD PBMC Decreased [28] miR-140, -182 CAD Whole blood Decreased [29] miR-122, -370 CAD Plasma Increased [21] miR-181a CAD Monocytes Decreased [30] Let-7i CAD Monocytes Decreased [31] miR-340, -624 CAD Platelets Increased [32]

miR-17, -21, -20a, -22a, -27a, -92a, -126, -145, -155, -221, -130a, -208b, let-7d miR-133a, -208a

miR-20b, -21, -24, -29b, -15a, -126, -150, -191, -197, -223, -320, -486 miR-28-3p

miR-188-5p, -135\*, -323-3p, -509-3p, -520-3p, -572, -573, 629\*, -632 miR-24, -106a, -191, -218, -222, -223, -342-3p, -412, let-7p

**Table 1.** Circulating miRNA in diseases associated with vascular calcification.

**Reference number**

127

[14]

[33]

[38]

Decreased

Role of MicroRNAs in Cardiovascular Calcification

http://dx.doi.org/10.5772/55326

Increased

Decreased

Increased

Decreased

Increased

The high incidence of cardiovascular complications in patients with CRD is partly explained by more aggressive development of atherosclerotic lesions and accelerated calcification [40]. To our knowledge, only one study reports circulating miRNA in patients with CRD. Neal *et*

tin in T2D animals.[39].

cell; HDL, high-density lipoprotein.


CRD, chronic renal disease; T2D, type 2 diabetes; CAD, coronary artery disease; AS, aortic stenosis; HC, familial hyper‐ cholesterolemia; AO, arteriosclerosis obliterans; PBMC, peripheral blood mononuclear cell; EPC, endothelial progenitor cell; HDL, high-density lipoprotein.

**Table 1.** Circulating miRNA in diseases associated with vascular calcification.

pared with the arteria mammaria interna as control tissue [17]. The healthy vessel ex‐ pressed higher levels of miR-520b and miR-105, whereas miR-10b, miR-218, miR-30e, miR-26b, and miR-125a were predominantly expressed in atherosclerotic plaque [17]. The investigators in both studies, however, did not examine miRNAs in calcified lesions. Microcalcification is thought to cause plaque rupture [18, 19]. Destabilized human pla‐ ques are characterized by a specific miRNA expression profile (high levels of miR-100, miR-127, miR-145, miR-133a, miR-133b). Target genes of these miRNAs (Nox1, MMP9, CD40) may play a role in vascular calcification [7]. Thus, miRNAs could participate in the formation of hydroxyapatite crystals, and thereby have an important role in regulating

Fichtlscherer *et al.* authored the first study investigating circulating miRNA in CAD [14]. Plasma levels of miR-17, miR-92a, miR-126, miR-145, and miR-155 were reduced in CAD compared to healthy controls, whereas miR-133a and miR-208a were increased [14]. Another study demonstrated a positive correlation of plasma miR-122 and miR-370 levels with the presence and severity of CAD [21]. Both miRNAs were significantly increased in hyperlipi‐ demia patients, compared to controls [21]. Increased levels of miR-27b, miR-130a, and miR-210

Comparison of published studies is challenging mainly because of the different sources of circulating miRNAs, which include serum, whole blood, PBMCs, EPCs, and platelets (Table 1). The miRNA profiles obtained from the different studies, therefore, are often not the same. In this context, a recent report suggested the necessity of careful selection for reference miRNAs by showing that hemolysis may significantly affect the levels of plasma miRNAs previously

Polymorphisms in the 3'UTR may alter miRNA binding, leading to post-transcriptional dysregulation of the target gene and aberrant protein level. Functional single-nucleotide polymorphisms (SNPs) of miRNA-binding sites associate with the risk of cardiovascular disease. Wu *et al.* discovered a SNP in the miR-149 binding site of the 5,10-methylenetetrahy‐ drofolate reductase (MTHFR) gene that associated with increased risk for CAD [23]. Further‐ more, a larger study in a Chinese population of 956 CAD patients and 620 controls revealed that a SNP in the binding sites for miR-196a2 and miR-499 associated with the occurrence and

T2D is a major risk factor for cardiovascular disease. Zampetaki *et al*. identified a plasma miRNA signature for T2D that includes reduced levels of miR-223, miR-15, miR-20b, miR-21, miR-24, miR-29b, miR-126, miR-150, miR-191, miR-197, miR-320, and miR-486, and elevated levels of miR-28-3p [33]. Reduced miR-126 levels antedated diabetes manifestation, and might explain the impaired peripheral angiogenic signaling in patients with T2D. Reduction of circulating miR-21 and miR-126 was confirmed by Meng *et al.*, who also found a decrease of miR-27a,b and miR-130a in T2D patients [35]. Another study demonstrated mostly elevated miRNA levels (miR-9, miR-29a, miR-30d, miR-34a, miR-124a, miR-146a, and miR-375) in serum from T2D patients, compared with pre-diabetic and/or normal glucose tolerance

atherosclerotic plaque toward unstable phenotypes and rupture [20].

were observed in the serum of arteriosclerosis obliterans patients [15].

**3.2. miRNAs in diabetes and chronic renal disease (CRD)**

used as controls [22].

126 Calcific Aortic Valve Disease

prognosis of CAD [24].

conditions [36]. In contrast, reduced miR-146a levels in PMBCs from Asian Indian T2D patients associated with insulin resistance, poor glycemic control, and several proinflammatory cytokine genes [34]. miR-146a participates in the transcriptional circuitry regulating fibronec‐ tin in T2D animals.[39].

The high incidence of cardiovascular complications in patients with CRD is partly explained by more aggressive development of atherosclerotic lesions and accelerated calcification [40]. To our knowledge, only one study reports circulating miRNA in patients with CRD. Neal *et* *al.* found that plasma levels of total and specific miRNAs (miR-16, miR-21, miR-155, miR-210, and miR-638) are reduced in CRD patients, compared to patients with normal renal function [37]. A strong correlation exists between detected circulating miRNAs and estimated glomer‐ ular filtration rate [37]. Interestingly, miR-638 was the only miRNA that showed a differential urine excretion in CRD patients [37]. Transforming growth factor beta (TGF-β), a pro-fibrotic key mediator of CRD, reduces levels of miR-192 [41] and miR-29a [42] and increases miR-377 levels [43] *in vitro* and *in vivo*, thereby promoting the expression of extracellular matrix components.

#### **3.3. miRNAs and aortic valve disease**

Aortic stenosis (AS) is typically caused by calcific aortic valve disease. To our knowledge, no study to date describes a specific miRNA signature in the circulation of patients with AS. Nigam *et al*. identified a miRNA pattern specific to AS using tissue from whole bicuspid valves and linking them to calcification-related genes, such as Smad1/3, Runx2, and BMP2 [44]. miR-26a, miR-30b, and miR-195 were decreased in the aortic valves of patients requiring replacement due to AS, compared to those requiring replacement due to aortic insufficiency [44]. Another group compared bicuspid with tricuspid aortic valve leaflets by miRNA microarray, and found a number of modulated miRNAs [45]. Particularly, miR-141 had the most dramatic change, showing a 14.5-fold decrease in the bicuspid versus tricuspid valve tissue, while the levels of calcification were comparable between the two groups.

#### **3.4. Similar miRNA profiles may represent common miRNAs in diseases associated with cardiovascular calcification**

Our detailed investigation using currently published literature revealed common circulat‐ ing miRNAs in diseases associated with vascular calcification. Seven miRNAs (miR-21, miR-27, miR-34a, miR-126, miR-146a, miR-155, and miR210) were useful biomarkers in atherosclerosis, T2D, and/or CRD, and only miR-21 was common among all three diseas‐ es [14, 33, 37] (Table 3).

Atherosclerotic arteries [16] and sclerotic intima from lower-extremity vessels [15] expressed higher miR-21 levels than did healthy vessels. Circulating levels of miR-21 in atherosclerosis, T2D, and/or CRD were reduced [14, 33, 37]. The reason for this discrepancy is unknown, and requires further investigation.

miR-146a is an inflammation-related miRNA, implicated in atherosclerosis and osteoclasto‐ genesis [46]. Circulating miR-146a is increased in CAD patients [25] and T2D [36]. In addition, miR-146a was more highly expressed in atherosclerotic arteries in an animal model [16], and associated with CRD *in vivo* [47]. miR-155, another inflammation-associated miRNA, is decreased in CAD [14] and CRD [37]. Deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability [48], suggesting that it acts as an anti-inflamma‐ tory and atheroprotective miRNA. miR-155 is also highly expressed in endothelial cells (ECs) and SMCs, where it targets angiotensin-II receptor [49]. The renin–angiotensin system participates in cardiovascular calcification [50, 51]. Angiotensin-receptor blockade can inhibit

arterial calcification by disrupting vascular osteogenesis *in vivo* [52]. In addition, an observa‐ tional study showed reduced progression of AV disease in patients taking angiotensinconverting enzyme inhibitors [53]. Furthermore, miR-155 represses osteoblastogenesis by targeting Smad proteins [54]. Thus, high expression of miR-155 may prevent cardiovascular

miR-155 ↓ miR-155 ↓

**CAD T2D CRD** miR-21 ↓ miR-21 ↓ miR-21 ↓

**miRNA Disease Tissue type Finding**

Atherosclerotic arteries

Atherosclerotic carotid artery

Sclerotic intima from lower extremities vessels

AS Bicuspid aortic valve

valves

CAD Destabilized plaque Increased [20]

CAD

CAD

AO

miR-26a, -30b, -195 AS Whole bicuspid

CAD, coronary artery disease; AS, aortic stenosis; AO, arteriosclerosis obliterans.

miR-27 ↓ miR-27 ↓ miR-34a ↑ miR-34a ↑ miR-126 ↓ miR-126 ↓

CRD, chronic renal disease; T2D, type 2 diabetes; CAD, coronary artery disease

**Table 3.** Common circulating miRNA in diseases associated with vascular calcification.

miR-21, -34a, -146a, -146b-5p, -210

miR-105, -520b miR-10b, -26b, -30e, -125a, -218,

> miR-100, -127, -133a,b -145

miR-221, -222 miR-21, -27b, -210, -130a, let-7f

miR-22, -27a, -141, -124, -125b, -185, -187, -194, -211, -330, -370, -449, -486, -551, -564, -575, -585, -622, -637, -648, -1202, -1282, -1469, -1908, -1972 miR-30e, -32, -145, -151, -152, -190, -373, -768

**Table 2.** miRNAs expressed in human calcified tissue.

**Reference number**

129

[17]

[15]

[45]

Increased [16]

http://dx.doi.org/10.5772/55326

Decreased

Role of MicroRNAs in Cardiovascular Calcification

Increased

Decreased

Increased

Decreased

Increased

Decreased [44]


CAD, coronary artery disease; AS, aortic stenosis; AO, arteriosclerosis obliterans.

**Table 2.** miRNAs expressed in human calcified tissue.

*al.* found that plasma levels of total and specific miRNAs (miR-16, miR-21, miR-155, miR-210, and miR-638) are reduced in CRD patients, compared to patients with normal renal function [37]. A strong correlation exists between detected circulating miRNAs and estimated glomer‐ ular filtration rate [37]. Interestingly, miR-638 was the only miRNA that showed a differential urine excretion in CRD patients [37]. Transforming growth factor beta (TGF-β), a pro-fibrotic key mediator of CRD, reduces levels of miR-192 [41] and miR-29a [42] and increases miR-377 levels [43] *in vitro* and *in vivo*, thereby promoting the expression of extracellular matrix

Aortic stenosis (AS) is typically caused by calcific aortic valve disease. To our knowledge, no study to date describes a specific miRNA signature in the circulation of patients with AS. Nigam *et al*. identified a miRNA pattern specific to AS using tissue from whole bicuspid valves and linking them to calcification-related genes, such as Smad1/3, Runx2, and BMP2 [44]. miR-26a, miR-30b, and miR-195 were decreased in the aortic valves of patients requiring replacement due to AS, compared to those requiring replacement due to aortic insufficiency [44]. Another group compared bicuspid with tricuspid aortic valve leaflets by miRNA microarray, and found a number of modulated miRNAs [45]. Particularly, miR-141 had the most dramatic change, showing a 14.5-fold decrease in the bicuspid versus tricuspid valve

tissue, while the levels of calcification were comparable between the two groups.

**3.4. Similar miRNA profiles may represent common miRNAs in diseases associated with**

Our detailed investigation using currently published literature revealed common circulat‐ ing miRNAs in diseases associated with vascular calcification. Seven miRNAs (miR-21, miR-27, miR-34a, miR-126, miR-146a, miR-155, and miR210) were useful biomarkers in atherosclerosis, T2D, and/or CRD, and only miR-21 was common among all three diseas‐

Atherosclerotic arteries [16] and sclerotic intima from lower-extremity vessels [15] expressed higher miR-21 levels than did healthy vessels. Circulating levels of miR-21 in atherosclerosis, T2D, and/or CRD were reduced [14, 33, 37]. The reason for this discrepancy is unknown, and

miR-146a is an inflammation-related miRNA, implicated in atherosclerosis and osteoclasto‐ genesis [46]. Circulating miR-146a is increased in CAD patients [25] and T2D [36]. In addition, miR-146a was more highly expressed in atherosclerotic arteries in an animal model [16], and associated with CRD *in vivo* [47]. miR-155, another inflammation-associated miRNA, is decreased in CAD [14] and CRD [37]. Deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability [48], suggesting that it acts as an anti-inflamma‐ tory and atheroprotective miRNA. miR-155 is also highly expressed in endothelial cells (ECs) and SMCs, where it targets angiotensin-II receptor [49]. The renin–angiotensin system participates in cardiovascular calcification [50, 51]. Angiotensin-receptor blockade can inhibit

components.

128 Calcific Aortic Valve Disease

**3.3. miRNAs and aortic valve disease**

**cardiovascular calcification**

es [14, 33, 37] (Table 3).

requires further investigation.


**Table 3.** Common circulating miRNA in diseases associated with vascular calcification.

arterial calcification by disrupting vascular osteogenesis *in vivo* [52]. In addition, an observa‐ tional study showed reduced progression of AV disease in patients taking angiotensinconverting enzyme inhibitors [53]. Furthermore, miR-155 represses osteoblastogenesis by targeting Smad proteins [54]. Thus, high expression of miR-155 may prevent cardiovascular calcification by inhibiting the BMP signalling pathway or the renin–angiotensin system, making it a promising anti-calcification therapeutic target.

expression to prevent skeletal disorders [77]. Three of these miRNAs (miR-133a, miR-135a, and miR-218) are altered in cardiovascular diseases associated with vascular calcification [14, 17, 20, 28]. Klotho mutant mice, which display vascular calcification due to hyperphosphate‐ mia and through a Runx2-dependent mechanism [78], show overexpression of miR-135a (together with miR-762, miR-714, and miR-712) in the aortic media, which causes SMC calcification by disruption of Ca2+ transporters and increasing intracellular Ca2+ concentrations [79]. More recently, miR-204, another candidate of the Runx2-cluster, was found to contribute to SMC calcification *in vitro* and *in vivo* [80]. Downstream targets of Runx2 are bone-specific genes like osteopontin, osterix and osteocalcin, all present in the cardiovascular osteogenic cell phenotype [2, 81]. We recently demonstrated that miR-125b, which inhibits osteoblast differentiation [82] regulates the transition of SMCs into osteoblast-like cells partially by targeting the transcription factor osterix, providing the first miR-dependent mechanism in the progression of vascular calcification [83]. Additionally, miRNA-processing enzymes —

Role of MicroRNAs in Cardiovascular Calcification

http://dx.doi.org/10.5772/55326

131

Another potent regulator of vascular and valvular calcification is the BMP signaling pathway (reviewed in detail elsewhere [85]). BMP2 and BMP4 are potent osteogenic differentiation factors detected in calcified valve and atherosclerotic lesions [86-88]. BMPs elicit their effects through activation of receptor complex composed of type I and type II receptors and activate receptor-type–dependent and ligand-dependent Smad transcription factors, which modulate the expression of Runx2 [85]. MiR-26a, miR-135, and miR-155 were previously reported as Smad-regulating miRNAs related to osteoblastogenesis; they functionally repress osteoblast differentiation by targeting Smad1 and Smad5, respectively [54]. miR-155 is one of the circulating miRNAs that is decreased in CAD [14] and CRD [37] (discussed earlier). miR-26a was repressed in aortic valve leaflets of patients with aortic stenosis, and human aortic valvular interstitial cells showed decreased mRNA levels of BMP2 and Smad1 when treated with miR-26a mimic [44]. The same group found lower expression of miR-30b, which targets Smad1 and Smad3. Another group reported deceased miR-141 levels together with increased BMP2 levels in bicuspid versus tricuspid aortic valve leaflets, and showed *in vitro* that miR-141 represses the VIC response to calcification, in part through BMP2-dependent calcification [45]. Itoh *et al*. identified miR-141 as a pre-osteoblast differentiation-related miRNA, which modulated the BMP2-induced pre-osteoblast differentiation by direct translational repression

Activation of canonical wingless-type (WNT) signaling is crucial for osteoblast function [90] and for the programming of valvular and vascular cells during cardiovascular calcification [85]. Activation of the Wnt/β-catenin signaling pathway occurs in human calcified aortic valve stenosis [91], in LDL receptor (LDLR)-deficient mice [92, 93], and in osteogenic SMCs *in vitro* [94]. Dickkopf (Dkk)1 is an extracellular antagonist of the canonical Wnt signaling that plays a crucial role in bone remodeling by binding to and inactivating signaling from LDLR-related protein 5/6 [95, 96]. Dkk-1 may also play a role in vascular calcification. In CRD patients, Dkk1 serum levels correlated negatively with arterial stiffness [97]. Dkk-1 prevents warfarininduced activation of β-catenin, and osteogenic transdifferentiation of SMCs [98] and TNF αinduced induction of alkaline phosphatase activity [92]. Remarkably, two miRNAs targeting

essential for SMC function [84] — were reduced in calcified SMCs [83].

of Dlx5, a transcription factor for osterix [89].

In summary, a set of circulating miRNAs (consisting of miR-21, miR-27, miR-34a, miR-126, miR-146a, miR-155, and miR-210) is dysregulated in various pro-inflammatory diseases and may represent a miRNA signature for cardiovascular calcification. Of note, systemic and local inflammation paradoxically affects cardiovascular calcification and bone loss, which supports the concept of inflammation-dependent cardiovascular calcification previously proposed by our group and others [13, 40, 55-57].
