Circulating miRNA in diseases associated with vascular calcification.
With a growing older population, cardiovascular diseases are becoming an increasing economic and social burden in Western societies. Cardiovascular calcification is a major characteristic of chronic inflammatory disorders — such as chronic renal disease (CRD), type 2 diabetes (T2D), atherosclerosis and calcific aortic valve disease (CAVD) — that associate with significant morbidity and mortality. Cardiovascular calcification also associates with osteoporosis in humans and animal models [1, 2] — the so-called “calcification paradox” . The concept that similar pathways control both bone remodeling and vascular calcification is currently widely accepted, but the precise mechanisms of calcification remain largely unknown. Osteogenic transition of vascular smooth muscle cells (SMCs), valvular interstitial cells (VIC) or stem cells is induced by bone morphogenetic proteins, inflammation, oxidative stress, or high phosphate levels, and leads to a unique molecular pattern marked by osteogenic transcription factors . Loss of mineralization inhibitors, such as matrix γ-carboxyglutamic acid Gla protein (MGP) and fetuin-A also contribute to cardiovascular calcification. The physiological balance between induction and inhibition of calcification becomes dysregulated in CRD, T2D, atherosclerosis, and CAVD. Consequently, calcification may occur at several sites in the cardiovascular system, including the intima and media of vessels and cardiac valves .
The central role of miRNAs as fine-tune regulators in the cardiovascular system and bone biology has gained acceptance and has raised the possibility for novel therapeutic targets. Circulating miRNAs have been proposed as biomarkers for a wide range of cardiovascular diseases, but knowledge of miRNA biology in cardiovascular calcification is very limited.
2. Micro-RNA biology: Biosynthesis and function
Micro-RNAs (miRNAs) are a large class of evolutionarily conserved, small, endogenous, non-coding RNAs serving as essential post-transcriptional modulators of gene expression . miRNAs regulate biological processes by binding to mRNA 3’-untranslated region (UTR) sequences to attenuate protein synthesis or messenger RNA (mRNA) stability . Acting as genetic switches or fine-tuners, miRNAs are key regulators of diverse biological and pathological 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 exons or within intergenic regions .
miRNA biogenesis is shown in Figure 1. The transcription process is mediated by the RNA-polymerase II that produces long precursor RNAs known as “primary miRNA” (pri-miRNA) with a typical hairpin morphology . A nuclear endonuclease, called DROSHA, then crops the distal stem portion of pri-RNA obtaining shorter chains (pre-miRNA) . Pre-miRNA is transported to the cytoplasm by the nuclear receptor Exportin-5  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 . Mature miRNA interact with proteins like Argonaute endonuclease (Arg 2), in order to form the RNA-induced silencing complex (RICS), which directs mature miRNA towards the targeted mRNA and bind on their 3’ untranslated region (UTR) .
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].
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 . Atherosclerotic calcification occurs as a part of atherogenic progress in the vessel intima. Small hydroxyapatite mineral crystals (microcalcification) can be visualized in early lesions . Medial calcification occurs primarily in association with CRD and T2D, independently of hypercholesterolemia. Aortic valve calcification leads to impaired movement of aortic valve leaflets, and causes valve dysfunction . 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 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) . In addition, tissue levels of miRNAs were investigated.
3.1. miRNAs in coronary artery disease
Studies about miRNA expression in calcified vessels are rare. Li
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 .
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
|miR-17, -21, -20a, -22a, -27a, -92a, -126, -145, -155, -221, -130a, -208b, let-7d|
|miR-140, -182||CAD||Whole blood||Decreased|||
|miR-20b, -21, -24, -29b, -15a, -126, -150, -191, -197, -223, -320, -486|
|miR-21, -27a, b, -126, -130a||T2D||EPC||Decreased|||
|miR-9, -29a, -30d, -34a, -124a, -146a, -375||T2D||Serum||Increased|||
|miR-16, -21, -155, -210, -638||CRD||Plasma||Decreased|||
|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
|miR-21, -27b, -130a, -210||AO||Serum||Increased|||
3.2. miRNAs in diabetes and chronic renal disease (CRD)
T2D is a major risk factor for cardiovascular disease. Zampetaki
The high incidence of cardiovascular complications in patients with CRD is partly explained by more aggressive development of atherosclerotic lesions and accelerated calcification . To our knowledge, only one study reports circulating miRNA in patients with CRD. Neal
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
|miR-21, -34a, -146a,|
miR-10b, -26b, -30e, -125a, -218,
|CAD||Atherosclerotic carotid artery||Decreased|||
|miR-100, -127, -133a,b|
miR-21, -27b, -210, -130a, let-7f
|AO||Sclerotic intima from lower extremities vessels||Decreased|||
|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
|AS||Bicuspid aortic valve||Decreased|||
|miR-26a, -30b, -195||AS||Whole bicuspid valves||Decreased|||
3.4. Similar miRNA profiles may represent common miRNAs in diseases associated with cardiovascular calcification
Our detailed investigation using currently published literature revealed common circulating 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 diseases [14, 33, 37] (Table 3).
|miR-21 ↓||miR-21 ↓||miR-21 ↓|
|miR-27 ↓||miR-27 ↓|
|miR-34a ↑||miR-34a ↑|
|miR-126 ↓||miR-126 ↓|
|miR-155 ↓||miR-155 ↓|
Atherosclerotic arteries  and sclerotic intima from lower-extremity vessels  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 osteoclastogenesis . Circulating miR-146a is increased in CAD patients  and T2D . In addition, miR-146a was more highly expressed in atherosclerotic arteries in an animal model , and associated with CRD
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].
4. miRNA and osteogenesis in the vascular wall
Cardiovascular calcification is an active, cell-regulated process. Various studies provide evidence of phenotypic transition or transition/dedifferentiation of mature SMCs or VICs into an osteogenic phenotype — a key feature in cardiovascular calcification. In medial calcification, SMCs undergo dedifferentiation from a contractile to a pro-atherogenic synthestic phenotype, lose the expression of their marker genes, acquire osteogenic markers, and deposit a mineralized bone-like matrix. In valvular calcification, VICs can undergo the transition to osteoblast-like bone-forming cells . Recently, a novel concept emerged of circulating cells harboring osteogenic potential that can home to atherosclerotic lesions and contribute to intimal calcification [59, 60]. Comparing the sources of cells that contribute to atherosclerotic intimal calcification revealed that SMCs are the major contributors that reprogram its lineage towards osteochondrogenesis/blastogenesis; circulating bone marrow-derived cells, however, also contribute to early osteochondrogenic differentiation in atherosclerotic vessels . The master transcription factors, including Runx2/Cbfa1, Msx2, and Osterix, designate cells for osteoblast lineages through the induction of downstream genes such as alkaline phosphatase, osteopontin, and osteocalcin. Here we summarize miRNAs involved in SMC differentiation, as well as in osteogenesis, with targets involved in cardiovascular calcification.
The SMC phenotype is dependent on the miR-143/145 cluster [62-64]. Circulating miR-145 levels are reduced in CAD patients . miR-145 is one of the most recognized arterial miRNAs . Inhibition of miR-143/145 promotes a phenotypic switch to the synthetic, pro-atherogenic SMC state , including the inhibition of SMC marker-like alpha-smooth muscle actin and smooth muscle myosin heavy chain  — both diminished in osteogenic SMCs . miR-145 modulates SMC differentiation by targeting Krüppel-like factor 4 (KLF4) . KLF4 mediates high phosphate-induced conversion of SMCs into osteogenic cells . Conversely, miR-145-deficient mice  and overexpression of miR-145  both reduce neointima formation in vascular injury.
Similar to miR-145, miR-133 has a potent inhibitory role on the vascular SMC phenotypic switch . Runx2, a cell-fate determining osteoblastic transcription factor, is a target of miR-133 . Runx2 acts as a critical regulator of osteogenic lineage and a modulator of bone-related genes . Runx2 is essential and sufficient for regulating osteogenesis in SMC and VIC [73, 74, 75, 76]. Discovered in the bone biology field, a program of miRNAs controls Runx2 expression to prevent skeletal disorders . 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 hyperphosphatemia and through a Runx2-dependent mechanism , 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 . More recently, miR-204, another candidate of the Runx2-cluster, was found to contribute to SMC calcification
Another potent regulator of vascular and valvular calcification is the BMP signaling pathway (reviewed in detail elsewhere ). 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 . 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 . miR-155 is one of the circulating miRNAs that is decreased in CAD  and CRD  (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 . 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
Activation of canonical wingless-type (WNT) signaling is crucial for osteoblast function  and for the programming of valvular and vascular cells during cardiovascular calcification . Activation of the Wnt/β-catenin signaling pathway occurs in human calcified aortic valve stenosis , in LDL receptor (LDLR)-deficient mice [92, 93], and in osteogenic SMCs
The contribution of osteoclasts to cardiovascular calcification is still controversial . The observation of osteoclast-like cells in calcified atherosclerotic lesions suggested this bone-related cell is active in the vessel wall. The evidence was strengthened recently by Sun et al., who demonstrated the functional role of SMC-derived Runx2 promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells — suggesting that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process . The receptor activator of the nuclear factor-kappa B (NF-kappa B) ligand (RANKL)/osteoprotegerin (OPG) system controls proper osteoclastogenesis, and actes as a biomarker for CAD [107, 108].
Taken together, osteogenic processes in both bone and the cardiovascular system are tightly controlled by miRNAs (Figure 2). Further studies are needed to elucidate whether interplay of miRNAs could explain the bone-vascular axis “calcification paradox,” or whether they act independently in the calcifying vessel and bone.
5. Circulating miRNAs as biomarkers and extracellular communicators
miRNAs are present in blood (plasma, platelets, erythrocytes, nucleated blood cells) with high stability. miRNAs can circulate in extracellular vesicles , in a protein complex (Ago2), or in a lipoprotein complex (HDL) , which prevents their degradation. Depending on the size and type, extracellular vesicles are broadly classified as ectosomes (also called shedding microvesicles), exosomes, matrix vesicles (MVs), and apoptotic bodies. Ectosomes are large extracellular vesicles 50-1000 nm in diameter; exosomes are small membranous vesicles of endocytic origin, 40-100 nm in diameter; MVs are 30-300 nm in diameter, are produced by blebbing of the plasma membrane, and can calcify; and apoptotic bodies, 50-5000 nm in diameter, are released from fragmented apoptotic cells.
The majority of miRNAs are independent of vesicles  and co-purify with the Ago2 complex [112, 113]. But in CAD patients, most plasma miRNAs associate with extracellular vesicles, and only a small amount are found in extracellular vesicle-free plasma . A cell-type-specific miRNA release and different export systems are implicated, as the miRNA release pattern within vesicles is different from that associated with Ago2 complexes . Thus, cells can select miRNA and pre-miRNA for controlled cellular release [115, 116]. miRNA profiles of extracellular vesicles are different from their maternal cell profiles, indicating an active mechanism of selective miRNA packing from cells into vesicles . We have limited knowledge about miRNA secretion. Blockade of sphingomyelinase inhibits exosome generation and miRNA secretion, and intercellular miRNA transfer implicates a ceramide-dependent mechanism [117, 118]. Ago2–miRNA complexes may be passively produced by dead cells, released by live cells, or actively transported though cell-membrane–associated channels or receptors .
Extracellular vesicles use miRNA to mediate intercellular communication over long distances or on a local tissue level . Endothelial apoptotic bodies can convey miR-126 to atherosclerotic lesions, which demonstrate uniquely paracrine-signaling function for miRNA during atherosclerosis [33, 121]. miRNA-containing vesicles can regulate intercellular communication between ECs and SMCs by selective packing of miR-143/145 in endothelial-derived vesicles, which are then transported to SMCs to control their phenotype .
How miRNAs are taken up by target cells and remain biologically active is still unknown. We know little about the mechanisms of vesicle-mediated cargo transfer. In physiological conditions, extracellular vesicles may bind to the membrane proteins of the surface of target cells through receptor–ligand interaction, resulting in intracellular stimulation of genetic pathways. They can also fuse with cell–target membranes and release genetic content in a nonselective manner. Furthermore, vesicles can bind to surface receptors on target cells with endocytotic internalization by recipient cells, followed by fusion with the membranes, leading to a release of their content into the cytosol of target cells .
A key event in the initiation and promotion of VIC and SMC calcification is the release of extracellular vesicles [81, 123]. Treatment of SMCs with elevated calcium levels promotes the production of calcifying vesicles (MVs), and the loss of fetuin-A, an inhibitor of mineral nucleation . These vesicles act as early nucleation sites for calcification. The phosphatidylserine-membrane complex from SMC-derived and macrophage-derived MVs redistributes and nucleates hydroxyapatite [125-127]. In addition, hydroxyapatite nanocrystals shed from vesicles may further promote mineralization via direct effects on SMC phenotype .
Insight into the underlying mechanism of selective packing of miRNAs into extracellular vesicles and selective uptake into the target cell will help increase understanding of the role of miRNA-containing vesicles in physiological intercellular communication, which may prevent calcification in the cardiovascular system.
6. miRNAs in the “calcification paradox”
Osteoporosis frequently associates with cardiovascular calcification, and the severity of aortic calcification associates positively with bone loss [2, 129, 130]. The “calcification paradox” could be explained by the shared molecular pathways in bone remodeling and cardiovascular calcification . How these two processes associate with each other and whether osteoporosis leads to cardiovascular calcification - or whether both disorders just share common risk factors - is unclear. In this section, we link cardiovascular calcification and bone loss and show commonalities in the systems’ miRNA pathways/patterns.
Studies of miRNA in patients with bone disease are lacking. A recent clinical study first reported miRNA as a potential biomarker for postmenopausal osteoporosis. Wang
Additionally, miR-2861 contributes to osteoporosis in mice and humans by targeting histone deacethylase 5, and thereby increasing Runx2 . No studies of miR-2861 in the cardiovascular system have been reported. Patients with rheumatoid arthritis also suffer from vascular calcification in different vessel beds, in addition to osteoporosis; the pathogenesis includes pro-inflammatory cytokines and site-specific inflammation (reviewed in detail elsewhere ). miR-146a, a negative regulator of inflammation and osteoclastogenesis, also associates with rheumatoid arthritis . Similar to patients with CAD, in patients with rheumatoid arthritis, miR-146a is up-regulated in PBMCs .
7. Conclusion and perspectives
Moreover, miRNA biology is very complex. Multiple miRNAs can target the same gene (e.g., Runx2–miRNA cluster), and one miRNA may have several targets. Only a small amount of these fine-tuned targets may alter biological responses and phenotypes. Understanding the role of miRNA in vascular calcification may be helpful in considering the paradoxical clinical observations of the concurrence of cardiovascular calcification and osteoporosis. Despite its global clinical burden, no medical therapies are available to treat cardiovascular calcification. Targeting of miRNA represents a novel therapeutic opportunity for treating calcification disorders. As vascular calcification and bone remodeling share common mechanisms, we have to understand in greater detail the functions of miRNAs and their association with the molecular pathogenesis of osteoporosis and vascular/valvular calcification.
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