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MicroRNAs in the Atherosclerotic Plaque.

According to the WHO, cardiovascular diseases (CVDs) (3) of atherosclerotic origin are the leading cause of death globally, accounting for 26.8% of deaths among men and for 31.5% among women. The occurrence of atherosclerosis begins to increase among middle-aged people. Autopsy studies have also shown that >80% of young adults who have died due to non-cardiac causes had a more than 25% stenosis in at least one of their main coronary arteries (1). Almost half of all patients suffer a major cardiovascular event, i.e., myocardial infarction or stroke, without a prior diagnosis of CVD. Sudden cardiac arrest accounts for 10% of total mortality and 40% of mortality from coronary heart disease. There are several patient characteristics and demographic and lifestyle factors, as well as clinical conditions, that are known risk factors for sudden cardiac arrest, but their combined predictive value is low. The addition of several lipid-related markers or their combinations to statistical models containing classical risk factors have led to only slight improvement in CVD prediction (2). Therefore, the recognition of high-risk cardiovascular patients remains one of the major unsolved problems in clinical practice.

MicroRNAs (miRNAs, miRs) are small [approximately 18-24 nucleotides (nt)] noncoding regulatory RNAs. They inversely regulate their target gene expression at the posttranscriptional level by inhibiting translation or causing degradation of the target messenger RNA (mRNA). The 18th release of the MicroRNA Database predicts that Homo sapiens may have 1527 miRNA precursors and 1921 mature miRNAs. The discrepancy between the number of mature miRNAs and precursors results from the fact that one miRNA precursor hairpin can be processed into several miRNAs (-3p and -5p or, for example, miR-21 and miR-21*). On the other hand, several distinct precursor miRNAs can be processed to form the same sequence, i.e., one mature miRNA. The number of predicted miRNAs in humans has increased steadily since the discovery of the molecules in humans in the early 21st century. It has also been predicted that the expression of more than one-third of human genes is regulated by one or more miRNAs, and the number of target genes is increasing in line with the discovery of novel miRNAs.

miRNAs are thought to be micromanagers of the expression of individual genes and biological pathways. Different cell types have different miRNA expression profiles, ranging from miRNAs expressed in most cell types to those that are preferably or even exclusively expressed in specific cell types. miRNAs have a crucial role in the development of animals, regulating the formation of tissues and organs (3). In addition, different pathological disease states have been shown to have identifiable miRNA profiles. Therefore, miRNA profiles could be used as diagnostic and prognostic markers for several diseases. This is possible because, unlike mRNAs, miRNAs are not rapidly degraded in plasma or other body fluids and can thus be detected reliably. They also may become useful in disease treatment because miRNA levels can be modified.

There are many good reviews describing the effects of miRNAs on cholesterol metabolism and human cardiac diseases. The rationale of the present review is to focus on the roles of miRNAs in the development of atherosclerotic lesions and in the changes that atherosclerosis may induce in the miRNA profile of different blood fractions. The present information originates from research done with cell cultures, mice, and humans, with special emphasis on the results obtained in studies using human samples. Our aim was also to elucidate the roles of these miRNAs, often connected to atherosclerosis only through in vivo and in vitro experiments, by relating their expression levels in situ to advanced atherosclerotic plaques and histologically healthy arterial tissue by using Tampere Vascular Study (TVS) data (4).

Atherosclerosis

Atherosclerosis is a systemic disease affecting the whole arterial tree of the human body. It can lead to multiple-site vascular disease (polyvascular disease) in the form of coronary artery, peripheral arterial, and cerebrovascular disease. Although cardiovascular deaths are caused by events in different areas of the arterial tree, a large proportion of them are caused by coronary artery disease (CAD). In addition to intravascular features, many systemic factors contribute to the risk of acute coronary events. Although atherosclerosis was at one time considered to be a lipid storage disease, subsequent research has shown that inflammatory cells, innate immunity, and inflammation play central roles in all stages of atherosclerotic disease from the early initial lesion to the late-stage plaque rupture (5).

The normal artery consists of 3 layers from the inner to the outer wall: the intima, media, and adventitia. Fatty streaks and lesions are found primarily in large and medium-sized muscular arteries in areas where the blood flow is turbulent or the velocity of the blood flow is decreased, leading to decreased levels of shear stress. The level of shear stress affects gene expression, signaling cascades, and cytokine secretion. Decreased endothelial shear stress also increases the intake, production, and oxidation of LDL in the fatty streaks of the arterial wall (6). Oxidized LDL (oxLDL) is cytotoxic, giving rise to a local inflammatory process which leads to the chronic inflammation observed in atherosclerosis and activation of endothelial cells. This inflammatory process increases the expression of adhesion molecules and infiltration of leukocytes. In the intima monocytes mature into macrophages, which engulf modified lipoproteins via receptor-mediated endocytosis. Subsequently, as a result of the accumulation of cholesterol, the macrophages transform into foam cells. As the fatty streaks progress into atherosclerotic lesions, an increased population of vascular smooth muscle cells (VSMCs) can be found in the thickened intima, underneath the layer of macrophages and foam cells. Activated VSMCs also produce components of the extracellular matrix involved in the evolution of the lipid-rich fatty streak into a more advanced fibrotic lesion. When the amount of extracellular lipids increases and a well-defined fibrous cap is formed, the lesion has developed into an atheroma.

The fibrous cap has an important role in maintaining the mechanical stability of the plaque. Steady growth of a stable atherosclerotic plaque usually does not lead to acute events until the stenosis becomes hemodynamically important. In most cases, end-organ ischemia and infarctions are caused by physical disruption of the lesion's fibrous cap, leading to release of thrombogenic constituents of the plaque and to the formation of a thrombus, which can block the blood flow in the artery. Alterations of the plaque surface (fissures and ulcerations) can show large variations between advanced atherosclerotic plaques (7). Studies have shown that plaque rupture is not a rare event in the progression of atherosclerosis and that only roughly 11% of discovered plaque ruptures are new, whereas most have been preceded by earlier, undiscovered ruptures.

Activated macrophages and T cells are frequently found at the site of the rupture. Inflammatory cells destabilize the plaque by secreting proinflammatory cytokines, proteases, coagulation factors, and vasoactive molecules (5). These molecules inhibit the formation of a stable fibrous cap, degrade the collagen in the cap, and initiate the formation of the clot. The stability of the plaque is also influenced by the cellular growth rate, apoptosis, and lipid metabolism of the vascular wall.

miRNA Biogenesis, Functions, and Transportation between Cells

miRNA BIOGENESIS

miRNAs are transcribed from the genome of the cell. The majority of mammalian miRNA genes are located in defined transcription units, and their expression is regulated by promoter areas similar to those found in protein-coding genes. Other miRNAs have been found in the introns and even exons of protein-coding genes (8). The expression of intragenic miRNAs is mostly regulated by the promoter of the host gene, which leads to similar expression patterns for the miRNA and the mRNA. Host gene-independent expression regulation of intragenic miRNAs has also been reported (9). The intergenic miRNAs have promoters of their own, which have the characteristics commonly associated with RNA polymerase Il-mediated transcription. Clustered miRNAs share one promoter and are co-regulated and transcribed as a long primary miRNA transcript (10). Most mammalian miRNAs are transcribed by RNA polymerase II, but some miRNAs, associated with short interspersed nuclear elements, and those originating from viruses are transcribed by RNA polymerase III.

Most miRNAs are transcribed as part of longer primary miRNA transcripts (pri-miRNAs) that are several kilobases long and capped, spliced, and polyadenylated. In such pri-miRNAs, the miRNA sequences form stem-loop structures (or hairpins). These stemloop structures are recognized by a multiprotein complex with Drosha and Di George syndrome critical region gene region 8 (DGCR8). The DGCR8 binds the double-stranded RNA, and the RNase III enzyme Drosha cleaves the double-stranded stem approximately 11 bp from the base of the stem and leaves a 2-nt overhang at the 3' end. The formed 70-100-bp-long RNA molecule is called precursor miRNA (premiRNA) (10). Another, rare biogenesis pathway has been indicated for miRNAs derived from introns (mintrons) that are similar in size as the premiRNAs to begin with and do not have to be processed by Drosha. The spliceosome splices mintrons out of the host gene transcripts to form looped intermediates (lariant), which then refold into the pre-miRNA structure (Fig. 1) (11).

Pre-miRNAs are recognized by the Exportin 5 protein and exported out of the nucleus through the nuclear pore complex in a RanGTP-dependent process. There, the pre-miRNAs are further processed by another RNase III enzyme, Dicer. This enzyme recognizes the 3' overhang of the pre-miRNA, binds it with its PAZ (Piwi, Argonaute, and Zwille) domain, and cleaves it into the mature miRNA. The processed miRNA is composed of double-stranded RNA overhangs with lengths of 22 bp and 2 nt on both ends. These mature miRNAs are incorporated into RNA-induced silencing complexes (RISC). While this complex assembles, the miRNA duplex is unwound by helicase and the passenger strand (miRNA*) is degraded, leaving the guide strand within the miRISC. Analysis has shown that the strand with less stable base pair binding in its 5' end is more often selected as a guide strand. It has been suggested that the Dicer is repositioned after the cleavage reaction and, in its new position, the helicase domain in Dicer senses the thermodynamic stability of the ends and incorporates the guide strand into the RISC (forming miRISC) (12).

FUNCTION OF miRNAS, miRNA TRANSPORTATION, AND SIGNALING BETWEEN CELLS AND TISSUES

The guide strand of the miRNA leads the miRISC to a partially complementary target mRNA. This complex binds to the target sequence that is more commonly located within the 3' untranslated region (UTR) of the mRNA (10). Functional miRNA target sequences in 5' UTR and in the open reading frame have also been reported (13). The seed region of the miRNA (the first 2-8 nt) is especially important for miRNA target recognition. Target prediction programs use these seed sequences to predict the mRNA targets for miRNAs, but the existence of a seed region binding sequence in mRNA does not guarantee miRNA binding or translational repression. Furthermore, due to the short seed region, single miRNAs have been predicted to bind hundreds of mRNAs, and individual mRNAs can also be bound by several miRNAs.

Different levels of complementarity between the miRNA and mRNA can lead to various effects on gene expression. If the complementarity is perfect, miRNA functions as a short interfering RNA (siRNA), and the target mRNA is sequence-specifically cleaved by the miRISC complex. This is rare in mammals, and most miRNAs bind their target with partial complementarity, leaving bulges in the formed double-stranded molecule (miRNA-mRNA). The bulges hinder the cleavage function of the miRISC, and this kind of binding leads to the repression of translation and degradation of the mRNA via deadenylation (10). The repression of translation has been hypothesized to be caused by the miRISC's interactions with the initiation of translation and by the steric hindrance of the translating ribosomes. The partially complementary binding of the miRISC can also lead to the recruitment of deadenylase complexes and to the removal or shortening of the poly(A) tail of the mRNA. The shortening or complete removal of the poly(A) tail induces the removal of the 5' cap of the mRNA. The uncapped mRNA is rapidly degraded by 5'-3' exoribonucleases such as 5'-3' exoribonuclease 1 (10).

miRNAs have also been suggested to have other roles in the cells, including functioning as translational activators (14) and possibly as transcriptional regulators. Some miRNAs have a nuclear localization sequence and are mainly located in the nucleus instead of the cytoplasm, where they could regulate gene transcription (15). miRNAs have also been localized to mitochondria, possibly regulating mitochondrial gene expression.

miRNAs can be transported between cells and tissues. Both membrane-free miRNAs and miRNAs associated with vesicles can be found in the blood. The nonvesicle-associated miRNAs are thought to be stabilized by protein complexes, such as the RISC proteins Argonaute 2 (16) and nucleophosmin 1 (17). The release mechanism of these miRNAs is unclear, but they may have been freed to the blood stream as a consequence of a passive release of the cell content in necrosis. Membrane-bound miRNAs have been found in apoptotic bodies, exosomes, and microvesicles. The packing of the miRNAs in these vesicles can be random, but a regulated method has also been suggested--for example, Zernecke et al. have shown that certain miRNAs are enriched in apoptotic bodies (18). miR143/145-enriched vesicles have been shown to convey atheroprotective signaling from endothelial cells to VSMCs (19). Circulating miRNAs have also been found in HDL particles. Cellular export of miRNAs to HDL has been shown to be regulated by neutral sphingomyelinase, and the delivery to receiving cells has been demonstrated to be dependent on scavenger receptor class B type I (20). Circulating miRNAs have been reported to be involved in cell-to-cell communication and, potentially, to have a role in disease progression (18).

miRNAs in Cardiovascular System Development and Vascular Biology

miRNAS IN CARDIOVASCULAR SYSTEM DEVELOPMENT

miRNAs are necessary for the proper development of the cardiovascular system. Dicer-deficient mice die during midgestation between E12.5 and E14.5. These embryos have severely compromised vascular development/maintenance in both the embryo and the yolk sack. More specifically, the deletion of Dicer expression in embryonic VSMCs leads to lethality and excessive bleeding. Furthermore, mouse embryos with cardiac-specific Dicer deletion die during gestation. These embryos exhibit pericardial edema and a poorly developed ventricular myocardium. In more detail, the miRNAs let-7f, miR-27b, -221, -222, -145, -143, -21, and the miR-17-92 cluster have been connected to the development of the vasculature. Similarly, miR-1, -133, -208a, -208b, and -499 have been shown to have a role in myocardial development. A detailed review of miRNAs in the development of the cardiovascular system has been provided by Boettger et al. (21).

miRNAS IN ARTERY WALL CELLS, CIRCULATING LEUKOCYTES, PERIPHERAL BLOOD FRACTIONS, AND ATHEROSCLEROTIC LESIONS

Endothelial cells. miR-21 has been shown to be upregulated in endothelial cells in response to shear stress (22), and miR-10a expression is decreased in atherosclerosis-prone areas (23). The expression of miR-155 has been thought to protect the endothelium by decreasing the expression of endothelin-1 and angiotensin II type I receptor (24). miR-126 has been shown to be specifically expressed in the endothelial cells, where it seems to modulate the phenotype of these cells and, particularly, the response to migration induced by vascular endothelial growth factor and fibroblast growth factor 2 (25). Interestingly, miR-126 has also been shown to regulate the expression of vascular cell adhesion molecule 1, an adhesion molecule that mediates leukocyte-endothelial cell adhesion in the arterial endothelium (26). In addition, apoptotic endothelial cells in atherosclerotic lesions have been shown to release miR-126 -enriched apoptotic bodies, which cause other endothelial cells to attract endothelial progenitor cells to the site, to hinder lesion development (18). In an atherosclerotic artery, miR-34a is thought to have a role in the apoptosis and senescence of endothelial cells (27), whereas the expression of miR-210 is induced by the hypoxia in the plaque and causes increased tubulogenesis of endothelial cells as well as possible neovascularization (28). Hypoxia has been demonstrated to increase lesion progression in atherosclerosis by promoting lipid accumulation, inflammation, and ATP depletion, and the neovascularization following the hypoxic state has been connected to rupture-prone plaques (Fig. 2).

VSMCs. miRNAs have also been found to have a role in determining the phenotype and modulating proliferation of VSMCs. The expression of miR-155 inhibits VSMC differentiation, possibly by decreasing angiotensin II type 1 receptor expression (29), whereas miR-21 stimulates their proliferation by affecting the expression of phosphatase and tensin homolog and Bcl-2 (B-cell CLL/lymphoma 2) (30). The apoptosis of VSMCs caused by reactive oxygen species (ROS) is also decreased as a consequence of miR-21 expression (31). miRNA-21 has been shown to participate in ROSinduced gene regulation and protection against apoptosis by hindering the translation of programmed cell death 4 (31). In addition, miR-146a promotes VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo by forming an expression feedback loop with Kruppel-like factor 4 and decreasing its expression (32). During the formation of an atherosclerotic plaque, some VSMCs undergo a phenotypic change from a contractile to a secreting phenotype. The secreting VSMCs produce extracellular matrix and metalloproteinases. All in all, miR-24 (33), -221 (34), -31 (35), -146a (32), -208 (36), and -26a (37) have been connected to the synthetic phenotype of VSMCs mainly functioning in platelet-derived growth factor signaling and the cell cycle. In contrast, miR-1 (38), -133 (39), -10a (40), -21 (30,41), -143, -145 (42), -100 (43), -204 (44), and let-7d (45) expression has been associated with the contractile VSMCs (Fig. 2), where they have been connected to the inhibition of cell proliferation and migration and promotion of contractility.

Leukocytes: lymphocytes, dendritic cells, and monocytes. The multiple roles of leukocytes in the development of atherosclerosis are thought to be regulated by miRNAs. Many miRNAs have been connected to the differentiation process of T cells [miR-150 (46), -125b (47), -182 (48), -146a (49), -29 (50), -326 (51)], and B cells [miR-150 (46), -185 (52)]. T-cell cytokine and chemokine production has an important role in the progression of the atherosclerotic plaque. Several cytokines are targeted by miR-155 in [CD4.sup.+] T cells, and mice with miR-[155.sup.-/-] are immunodeficient (53).miRNA-155 is also needed for the survival of regulatory T cells and for the T-helper 17 response (54). miRNAs may also affect T-cell function in atherosclerosis by regulating antigen-presenting cells--for example, dendritic cells. miR-155 and -146a in particular have been hypothesized to have an important role in dendritic cell function and activation (55).

The differentiation of monocytes to macrophages results in changes in the miRNA expression profile (Fig. 2). miRNA-17-5p, miR-20a, and miR-106a expression decreases, allowing their target Runt-related transcription factor 1 to be upregulated, which leads to increased colony stimulating factor receptor expression and monocyte differentiation (56). In the differentiation process, the expression of miR-21 (57) and miR-146 (58) increases. The polarization of macrophages has also been shown to alter miRNA profiles. The activation of macrophages (towards the M1 and/or M2 phenotype) was shown by Graff et al. to increase the expression of miR-125a, -193b, -27a *, -155 *, and -29b-1 * and to decrease the expression of miR-26a*. Interestingly, the expression of miR-222* was increased in M2 macrophages and decreased in M1 macrophages in their study (59). In contrast, Zhang et al. found miR-181a, -155-5p, -204-5p, and -451 to be significantly upregulated and miR-125-5p, -146a, 143-3p, and -145-5p to be significantly downregulated when these investigators compared murine M1 macrophages to M2 macrophages (60). When the differentiated macrophages are treated with oxLDL to stimulate foam cell formation, miR-146a, -146b-5p, -155, -9, and -125a-5p expressions are upregulated (61). Of these upregulated miRNAs, miR-155 functions as a negative feedback regulator, decreasing the inflammatory response and lipid uptake through scavenger receptors (62). In contrast to Chen et al. (62), Yang et al. found miR-146a to be significantly downregulated in oxLDL-stimulated macrophages. They also found this miRNA to have very similar functions as miR-155 after the stimulation, mainly decreasing cytokine production and lipid uptake (63). The results regarding miR-155 in macrophages, foam cells, and even in murine atherosclerotic plaques are conflicting. Both pro- and antiinflammatory effects have been reported (64, 65). Recent studies indicate that the oxidization level of LDL engulfed by macrophages may affect the changes of miR-155 expression (65) and that even in mice the level of dyslipidemia may determine whether this miRNA functions as an anti- or proatherogenic molecule (64).

Circulating miRNAs. Atherosclerosis may also exert an effect on the expression of miRNAs in the peripheral blood of the patients. miRNAs have been profiled from the erythrocytes, platelets, serum, and plasma, and the miRNA profiles of individuals with and without CVD have been shown to differ from each other (66). For example, Fichtlscherer et al. have demonstrated that miR-133 and -208a are upregulated and that miR-126, -17, -92a, -155, and -145 are downregulated in the serum and plasma of individuals with CAD (66). miRNA profiling from plasma and serum should be viewed carefully, because blood clotting and sample preparation, for example, affect the miRNA profiles in these blood fractions (67). Hoekstra et al. have reported, in the same setting, that miR-135a, -134, -198, and -370 are upregulated and miR-147 is downregulated in the blood peripheral mononuclear cells (PBMCs), i.e., lymphocytes, monocytes, and macrophages (68). Li et al. indicated that they can identify individuals with stable CAD, acute coronary syndrome, and no CAD by the miR-146a profile in their PBMCs (69). Furthermore, the researchers from Iwate Medical School have reported the upregulation of miR-146a and -146b (70) as well as the downregulation of let-7i (71) in the PBMCs of CAD patients compared with non-CAD patients. In addition, Sondermeijer et al. have reported the upregulation of miR-340* and miR-624 * in the platelets of individuals with premature CAD, postulating that miRNAs in platelets can potentially fine-tune the gene expression involved in platelet reactivity and could thereby affect platelet-related atherothrombotic CVDs (72). A recently reported study by Vickers et al. shows that miRNAs also can be transported by lipoproteins and that familial hyperlipidemia can alter this profile. Their findings also indicated that other lipoproteins besides HDL, such as LDL, may also facilitate miRNA transport (20), thereby making the whole lipoprotein system a part of the miRNA signaling system, mediating its effects between different cells and tissues via the blood and/or extracellular fluids.

miRNAs in the atherosclerotic plaque: a summary of the expression of the vascular biology-associated miRNAs discussed in this review in atherosclerotic plaques found in the TVS. The miRNA expression profile has not been studied extensively in situ. The miRNA expression profile of carotid neointimal lesions in rats has been described. In this animal study, comparison of a neointimal lesion to a healthy artery showed that miR-21 is the most upregulated miRNA (30). Furthermore, Li et al., while studying the expression of 13 preselected miRNAs, observed increased expression of miR-21, -130a, -27b, -210, and let-7f in the intimal layer of arterial plaques from patients with atherosclerosis. These investigators also showed a parallel upregulation of miR130a, miR-27b, and miR-210 in the serum of patients with atherosclerosis (73). The upregulation of miR30e-5p, -26b, and -125a, and the downregulation of miR-520b and miR-105 have also been reported in carotid plaques in comparison to nonatherosclerotic left internal thoracic arteries (LITA) (74), and Cipollone et al. found that, from a preselected pool of 41 miRNAs, miR-100, -127, -133a, -133b, and -145 were significantly upregulated in symptomatic carotid plaques when compared to asymptomatic plaques (75).

We have previously published the miRNA expression profiles of human atherosclerotic plaques from peripheral arteries (carotid, femoral, and abdominal aorta) in comparison to LITAs (4). We found that miR-21, -34a, -146a, -146b-5p, and -210 were the most upregulated miRNAs in atherosclerotic plaques in comparison to LITAs. We also found several predicted targets of these miRNAs to be downregulated in plaques, connecting them to the morphology, phenotype, and proliferation of VSMC as well as HDL and LDL metabolism.

Further utilizing our miRNA profiling from the peripheral atherosclerotic plaques in comparison to healthy LITAs, we have analyzed the expressions of the miRNAs presented in this review (Table 1). This analysis shows that several miRNAs previously found to be expressed differently in the blood fractions of CAD patients vs non-CAD individuals also demonstrate similar expression differences in atherosclerotic plaques in at least one vascular bed compared with a healthy arterial wall (miR-126, -134, -145, -146a, -198, -210, -340*, and -92a). Moreover, several miRNAs that have been connected to atherosclerosis by cell culture experiments have been found to be expressed in a similar pattern in the plaque, as predicted by these experiments. For example, those miRNAs that have been shown to have an increased expression in the development of monocytes to macrophages and foam cells were often upregulated in the plaque (miR-146a, -146b-5p, -155, -21), and several miRNAs indicated in the contractive phenotype of VSMCs were also down-regulated (miR-10a, -133, -145), whereas those connected to the secreting phenotype were upregulated in the plaque tissue compared with healthy arteries (miR-146a, -21, -221). The complexity of the atherosclerotic plaque composition and formation process and the multiple roles of miRNAs in tissue may partly explain why this kind of clear connection cannot be seen with all the miRNAs.

The functions as well as cell- and tissue-specific expression sites of all the miRNAs that have been discussed in this review are summarized and related to their expressions in atherosclerotic plaques from different arterial beds as determined in the TVS (Table 1) (4). This information may provide a more systematic insight into the function of miRNAs in human atherosclerotic lesions.

Clinical Implications

miRNA profiling can provide additional information about the biological processes involved in atherosclerosis, but miRNAs have also been thought to be potential biomarkers and drug targets. Circulating miRNAs have many qualities that make them attractive candidate molecular biomarkers. They are stable and evolutionarily conserved, and the changes in their expression are often tissue or disease specific. They are found in many body fluids such as urine, plasma, serum, and cerebrospinal fluid. Quantitative PCR assays can also make miRNA detection sensitive and specific (76). A drawback is that the normalization of miRNA expression has not yet been standardized, and the effects of blood sample collection and the form of the sample (e.g., serum or plasma) on the miRNA profile in the blood fraction must be taken into an account and optimized carefully (67). Moreover, the influences of age, health status, and dynamic changes of the circulating miRNA profile in different individuals have not been studied sufficiently.

Most of the miRNA biomarker research has been conducted in the context of cancer. Currently, several biomarkers for clinical diagnosis have been marketed. Prometheus Laboratories and Rosetta Genomics have launched a laboratory test intended to discover the source of cancer metastasis by its miRNA profile. Asuragen has also developed tests to diagnose pancreatic cancer (77). Efforts to identify miRNAs whose expression in the blood reflects the current status or future progression of atherosclerotic plaques are ongoing, as we reviewed in the circulating miRNA section (20, 66, 68, 72). So far, only the upregulation of miR-146a in the PBMCs of individuals with CAD in comparison to those not suffering from CAD has been replicated, in 2 independent studies (69, 70).

miRNAs have also been seen as direct drug targets. The first locked-nucleic-acid-based drug used in the treatment of hepatitis C has entered phase 2 clinical trials (78). Interesting drug targets may be miRNAs related to early endothelial dysfunction, such as miR-10a (23) and miR-126 (25). It maybe possible to stabilize an unstable plaque by affecting the expression of the miRNAs associated with the rupture-prone phenotype (75), as Lovren et al. have shown by promoting the contractile phenotype of VSMCs by the overexpression of miR-145 (79).

Major cardiovascular events are currently prevented by the treatment of risk factors. Most importantly, statins have become widely used for lowering LDL concentrations. Statins have been shown to decrease the expression of miR-146a and -146b (70) and to increase let-7i (71) expression in the PBMCs of CAD patients. More specifically, atorvastatin has been demonstrated to decrease miR-221/222 (80) and miR-34a (81) in circulating endothelial progenitor cells, whereas pravastatin or rosuvastatin did not have this effect. The divergent effects of different statins on miRNA expression might be related to the suggested pleiotropic effects of statins. Treatment that directly inhibits miR-33a/b expression has been shown to raise plasma HDL and to lower VLDL triglycerides in African green monkeys (82). Altered miRNA profiles have been associated with other CVD risk factors such as hypertension, diabetes, and smoking, but more research is needed to elucidate the clinical potential of these profiles.

Conclusions and Future Perspectives

Processes related to atherosclerosis have been shown to affect the miRNA expression in cell cultures and animal model experiments. Recently, there also has been active research on biomarker miRNAs for CVDs from different blood fractions. Few reports are available concerning the miRNA expression in actual human atherosclerosis, and the function of miRNAs in human atherosclerotic arteries remains largely unknown. We have reviewed miRNA expression studies on different blood fractions and cells closely related to atherosclerosis and elucidated how these miRNAs, predicted to have a role in human atherosclerosis, are expressed in advanced atherosclerotic lesions in comparison with healthy arteries. Many of the miRNAs shown to have altered expression profiles in the blood fractions of individuals with CVD are also dysregulated in the atherosclerotic artery wall. Such information suggests that the expression of these markers may reflect the changes actually taking place in the arterial wall. We also found that many miRNAs connected to atherosclerosis in cell culture studies are also expressed in the plaque in the same fashion as the experiments have predicted. For example, mir-126, -145, -146a, and -210 are expressed similarly in several tissues and cell types related to athero-sclerosis, providing evidence for an association with CVDs. As miRNA in atherosclerosis is still a relatively new field of research, the full clinical potential of these small RNAs in the diagnostics and treatment of CVDs remains to be elucidated.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: European Union 7th Framework Programme funding for the AtheroRemo project, 201668; E. Raitoharju, the Foundation of Clinical Chemistry, the Finnish Cultural Foundation, the Aarne Koskelo Foundation, the Tampere City Science Foundation, and the Alfred Kordelin Foundation; N. Oksala, the Emil Aaltonen Foundation; T. Lehtimaki, the Finnish Foundation of Cardiovascular Research, the Tampere Tuberculosis Foundation, the Tampere University Hospital Medical Fund grants 9M048 and 9N035, and the Emil Aaltonen Foundation.

Expert Testimony: None declared.

Patents: None declared.

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Emma Raitoharju, [1] * Niku Oksala, [1,2] and Terho Lehtimaki [1]

[1] Department of Clinical Chemistry, Pirkanmaa Hospital District, Fimlab Laboratories and University of Tampere, School of Medicine, Finland; [2] Division of Vascular surgery, Department of Surgery, Tampere University Hospital, Finland.

[3] Nonstandard abbreviations: CVD, cardiovascular disease; miRNA, miR, microRNA; nt, nucleotide; mRNA, messenger RNA; CAD, coronary artery disease; TVS, Tampere Vascular Study; oxLDL, oxidized LDL; VSMC, vascular smooth muscle cell; pri-miRNA, primary miRNA; DGCR8, Drosha and Di George syndrome critical region gene region 8; pre-miRNA, precursor miRNA; PAZ, Piwi, Argonaute, and Zwille; RISC, RNA-induced silencing complexes; UTR, untranslated region; siRNA, short interfering RNA; VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; PBMC, blood peripheral mononuclear cells; LITA, left internal thoracic artery.

* Address correspondence to this author at: Clinical Chemistry, Pirkanmaa Hospital District, Fimlab Laboratories and University of Tampere, School of Medicine, Biokatu 8, 33520 Tampere, Finland. Fax +358-331174168; e-mail emma.raitoharju@uta.fi.

Received February 6, 2013; accepted May 9, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.204917

Table 1 miRNAs associated with CVD as discussed in this review. (a,b)

                    In previously studied atherosclerosis-related
                                   tissue or cell type

miRNA             Serum (c)          Plasma (c)        Platelets (c)

let-7d
let-7f
let-7i
miR-1
miR-100
miR-105
miR-106a
miR-10a
miR-125a-5p
miR-125b
miR-126       [down arrow] (66)   [down arrow] (66)
miR-127
miR-130a       [up arrow] (73)
miR-133
miR-133a       [up arrow] (66)     [up arrow] (66)
m iR-133b
miR-134
miR-135a
miR-143
m iR-145      [down arrow] (66)   [down arrow] (66)
miR-146
miR-146a
miR-146b-5p
m iR-147
miR-150

miR-155       [down arrow] (66)   [down arrow] (66)

miR-155 *
miR-17        [down arrow] (66)   [down arrow] (66)
miR-17-92
miR-17-5p
miR-181a
miR-182
miR-185
miR-193b
miR-198
miR-204
miR-208
miR-208a       [up arrow] (66)     [up arrow] (66)
miR-20a
miR-21

miR-210        [up arrow] (73)
miR-221
miR-222 *

miR-24
mir-26a
miR-26a *
miR-26b
miR-27a *
miR-27b        [up arrow] (73)
miR-29
miR-30e-5p
miR-31
miR-326
miR-340*                                              [up arrow] (72)
miR-34a
miR-370
miR-451
miR-520b
miR-624 *                                             [up arrow] (72)
miR-9
miR-92a       [down arrow] (66)   [down arrow] (66)

              In previously studied atherosclerosis-related
                          tissue or cell type

miRNA              PBMCs (c)           B cell (c)

let-7d
let-7f
let-7i         [down arrow] (70)
miR-1
miR-100
miR-105
miR-106a
miR-10a
miR-125a-5p
miR-125b
miR-126
miR-127
miR-130a
miR-133
miR-133a
m iR-133b
miR-134         [up arrow] (68)
miR-135a        [up arrow] (68)
miR-143
m iR-145
miR-146
miR-146a      [up arrow] (69, 70)
miR-146b-5p
m iR-147       [down arrow] (68)
miR-150                             [down arrow] (46)

miR-155

miR-155 *
miR-17
miR-17-92                            [up arrow] (84)
miR-17-5p
miR-181a
miR-182
miR-185                             [down arrow] (52)
miR-193b
miR-198         [up arrow] (68)
miR-204
miR-208
miR-208a
miR-20a
miR-21

miR-210
miR-221
miR-222 *

miR-24
mir-26a
miR-26a *
miR-26b
miR-27a *
miR-27b
miR-29
miR-30e-5p
miR-31
miR-326
miR-340*
miR-34a
miR-370         [up arrow] (68)
miR-451
miR-520b
miR-624 *
miR-9
miR-92a

              In previously studied atherosclerosis-related
                          tissue or cell type

miRNA            T cell (d)             Monocyte (e)

let-7d
let-7f
let-7i
miR-1
miR-100
miR-105
miR-106a                              [down arrow] (83)
miR-10a
miR-125a-5p                         [up arrow] (59) (M1)
miR-125b       [up arrow] (47)    [down arrow] (6O) (M1/M2)
miR-126
miR-127
miR-130a
miR-133
miR-133a
m iR-133b
miR-134
miR-135a
miR-143                           [down arrow] (6O) (M1/M2)
m iR-145                          [down arrow] (60) (M1/M2)
miR-146                                [up arrow] (58)
miR-146a       [up arrow] (49)    [down arrow] (60) (M1/M2)
miR-146b-5p
m iR-147
miR-150       [down arrow] (46)

miR-155        [up arrow] (54)     [up arrow] (60) (M1/M2)

miR-155 *                          [up arrow] (59) (M1,2)
miR-17
miR-17-92      [up arrow] (84)
miR-17-5p                             [down arrow] (83)
miR-181a                           [up arrow] (6O) (M1/M2)
miR-182        [up arrow] (48)
miR-185
miR-193b                            [up arrow] (59) (M2)
miR-198
miR-204                            [up arrow] (6O) (M1/M2)
miR-208
miR-208a
miR-20a                               [down arrow] (83)
miR-21                                 [up arrow] (57)

miR-210
miR-221
miR-222 *                             [down arrow] (M1)
                                    [up arrow] (M2) (59)
miR-24
mir-26a
miR-26a *                          [up arrow] (59) (M1,2)
miR-26b
miR-27a *                           [up arrow] (59) (M2)
miR-27b
miR-29        [down arrow] (50)    [up arrow] (59) (M1,2)
miR-30e-5p
miR-31
miR-326        [up arrow] (51)
miR-340*
miR-34a
miR-370
miR-451                            [up arrow] (60) (M1/M2)
miR-520b
miR-624 *
miR-9
miR-92a

              In previously studied atherosclerosis-related
                          tissue or cell type

                                        Endothelial
miRNA          Macrophages (f)           cells (g)

let-7d
let-7f
let-7i
miR-1
miR-100
miR-105
miR-106a
miR-10a
miR-125a-5p    [up arrow] (61)
miR-125b
miR-126                            [down arrow] (25, 26)
miR-127

miR-130a
miR-133
miR-133a
m iR-133b
miR-134
miR-135a
miR-143
m iR-145
miR-146
miR-146a       [up arrow] (61)
miR-146b-5p    [up arrow] (61)
m iR-147
miR-150

miR-155       [up arrow] (61,65)     [down arrow] (24)
              [down arrow] (64)
miR-155 *
miR-17
miR-17-92
miR-17-5p
miR-181a
miR-182
miR-185
miR-193b
miR-198
miR-204
miR-208
miR-208a
miR-20a
miR-21                               [down arrow] (22)

miR-210                               [up arrow] (28)
miR-221
miR-222 *

miR-24
mir-26a
miR-26a *
miR-26b
miR-27a *
miR-27b
miR-29
miR-30e-5p
miR-31
miR-326
miR-340*
miR-34a                               [up arrow] (27)
miR-370
miR-451
miR-520b
miR-624 *
miR-9          [up arrow] (61)
miR-92a

              In previously studied atherosclerosis-related
                         tissue or cell type

                                       Atherosclerotic
miRNA               VSMCs (h)            plaque (i)

let-7d          [down arrow] (45)
let-7f                                 [up arrow] (73)
let-7i
miR-1           [down arrow] (38)
miR-100         [down arrow] (43)     [up arrow] (75) *
miR-105                               [down arrow] (74)
miR-106a
miR-10a         [down arrow] (40)     [down arrow] (23)
miR-125a-5p                            [up arrow] (74)
miR-125b
miR-126
miR-127                               [up arrow] (75) *
miR-130a                               [up arrow] (73)
miR-133         [down arrow] (39)
miR-133a                              [up arrow] (75) *
m iR-133b                             [up arrow] (75) *
miR-134
miR-135a
miR-143         [down arrow] (42)
m iR-145      [down arrow] (42, 79)   [up arrow] (75) *
miR-146
miR-146a         [up arrow] (32)
miR-146b-5p
m iR-147
miR-150

miR-155         [down arrow] (29)

miR-155 *
miR-17
miR-17-92
miR-17-5p
miR-181a
miR-182
miR-185
miR-193b
miR-198
miR-204         [down arrow] (44)
miR-208          [up arrow] (36)
miR-208a
miR-20a
miR-21         [up arrow] (30,31)      [up arrow] (73)
                [down arrow] (41)
miR-210                                [up arrow] (73)
miR-221         [down arrow] (34)
miR-222 *

miR-24           [up arrow] (33)
mir-26a          [up arrow] (37)
miR-26a *
miR-26b                                [up arrow] (74)
miR-27a *
miR-27b                                [up arrow] (73)
miR-29
miR-30e-5p                             [up arrow] (74)
miR-31           [up arrow] (35)
miR-326
miR-340*
miR-34a
miR-370
miR-451
miR-520b                              [down arrow] (74)
miR-624 *
miR-9
miR-92a

                  In the TVS, Raitoharju et al. (4)

                Carotid         Aortic        Femoral
miRNA          plaque (j)     plaque (j)     plaque (j)

let-7d
let-7f
let-7i                                       [up arrow]
miR-1
miR-100
miR-105
miR-106a
miR-10a       [down arrow]
miR-125a-5p
miR-125b
miR-126       [down arrow]
miR-127        [up arrow]
miR-130a
miR-133
miR-133a                     [down arrow]   [down arrow]
m iR-133b                    [down arrow]   [down arrow]
miR-134                                      [up arrow]
miR-135a
miR-143
m iR-145                     [down arrow]   [down arrow]
miR-146
miR-146a                      [up arrow]     [up arrow]
miR-146b-5p                   [up arrow]     [up arrow]
m iR-147
miR-150                       [up arrow]     [up arrow]

miR-155        [up arrow]     [up arrow]     [up arrow]

miR-155 *
miR-17
miR-17-92
miR-17-5p
miR-181a
miR-182       [down arrow]
miR-185       [down arrow]
miR-193b
miR-198                                      [up arrow]
miR-204
miR-208
miR-208a
miR-20a
miR-21         [up arrow]     [up arrow]     [up arrow]

miR-210        [up arrow]     [up arrow]     [up arrow]
miR-221                       [up arrow]     [up arrow]
miR-222 *

miR-24
mir-26a
miR-26a *
miR-26b
miR-27a *
miR-27b
miR-29
miR-30e-5p
miR-31
miR-326                                      [up arrow]
miR-340*                                     [up arrow]
miR-34a        [up arrow]     [up arrow]     [up arrow]
miR-370       [down arrow]
miR-451
miR-520b
miR-624 *
miR-9
miR-92a       [down arrow]

                 In the TVS, Raitoharju
                        et al. (4)

                Expressed       Expressed
               in healthy       in plaque
miRNA         arteries (k)     tissue (k)

let-7d             Yes             Yes
let-7f             Yes             Yes
let-7i             Yes             Yes
miR-1              Yes             Yes
miR-100            Yes             Yes
miR-105            No              No
miR-106a           No              No
miR-10a            Yes             Yes
miR-125a-5p        Yes             Yes
miR-125b           Yes             Yes
miR-126            Yes             Yes
miR-127            Yes             Yes
miR-130a           Yes             Yes
miR-133       Not available   Not available
miR-133a           Yes             Yes
m iR-133b          Yes             Yes
miR-134            Yes             Yes
miR-135a           No              No
miR-143            Yes             Yes
m iR-145           Yes             Yes
miR-146       Not available   Not available
miR-146a           Yes             Yes
miR-146b-5p        Yes             Yes
m iR-147           No              No
miR-150            Yes             Yes

miR-155            Yes             Yes

miR-155 *          No              No
miR-17             Yes             Yes
miR-17-92     Not available   Not available
miR-17-5p          Yes             Yes
miR-181a           Yes             Yes
miR-182            Yes             Yes
miR-185            Yes             Yes
miR-193b           Yes             Yes
miR-198            Yes             Yes
miR-204            Yes             Yes
miR-208       Not available   Not available
miR-208a           No              No
miR-20a            Yes             Yes
miR-21             Yes             Yes

miR-210            Yes             Yes
miR-221            Yes             Yes
miR-222 *          No              No

miR-24             Yes             Yes
mir-26a            Yes             Yes
miR-26a *          No              No
miR-26b            Yes             Yes
miR-27a *          No              Yes
miR-27b            Yes             Yes
miR-29             Yes             Yes
miR-30e-5p         Yes             Yes
miR-31             Yes             No
miR-326            Yes             Yes
miR-340*           Yes             Yes
miR-34a            Yes             Yes
miR-370            Yes             Yes
miR-451            Yes             Yes
miR-520b           Yes             Yes
miR-624 *          Yes             Yes
miR-9              No              No
miR-92a            Yes             Yes

(a) miRNAs are summarized and related to their expression in
atherosclerotic plaques in different arterial beds and in healthy
arteries included in the TVS. The arrows indicate whether the miRNA
has been up-or downreguiated as reported in the literature or in the
atherosclerotic plaques vs healthy arteries in the TVS.

(b) References referred to within this table: Weber et al. (22)] Fang
et al. (23); Zhu et al. (24); Fish et al. (25); FHarris et al. (26);
Ito et al. (27); Ivan et al. (28); Zheng et al. (29); Ji et al. (30);
Lin et al. (31); Sun et al. (32); Chan et al. (33); Davis et al.
(34); Liu et al. (35); Zhang et al. (36); Leeper et al. (37); Chen et
al. (38); Torella et al. (39); Fluang et al. (40); Kang et al. (41);
Cordes et al. (42); Grundmann et al. (43); Courboulin et al. (44);
Xiao et al. (45); Xiao et al. (46); Rossi et al. (47); Stittrich et
al. (48); Curtale et al. (49); Steiner et al. (50); Du et al. (51);
Belver et al. (52); Zernecke et al. (54); Kasashima et al. (57);
Taganov et al. (58); Graff et al. (59); Zhang etal. (60); Chen et al.
(61); Donnerset al. (64); Nazari-Jahantigh et al. (65); Fichtlscherer
et al. (66); Hoekstra et al. (68); Li etal. (69); Taka hash i et al.
(70); Sondermeijer et al. (72); Li etal. (73); Bidzhekov etal. (74);
Cipol lone et al. (75); Lovren et al. (79); Weber et al. (83); Xiao
et al. (84).

(c) In individuals with cardiovascular diseases vs individuals
without.

(d) In differentiated leucocytes vs nondifferentiated leucocytes.

(e) In macrophages vs monocytes; or (M1), in macrophage M1
activation; (M2), in macrophage M2 activation; (M1,2), in macrophage
M1 and M2 activation; or (M1/M2), in macrophage M1 activation in
comparison to M2 activation.

(f) In foam ceils vs macrophages.

(g) In endothelial cells subjected to atherosclerotic changes vs
native endothelial cells.

(h) In vascular smooth muscle cells with the synthetic vs contractile
phenotype.

(i) In atherosclerotic plaque vs healthy arteries, or *, in
rupture-prone atherosclerotic plaques vs stable plaques.

(j) In peripheral human atherosclerotic plaque vs healthy left
internal thoracic artery,

(k) miRNA was defined as expressed if it was expressed in at least
half of the plaque tissues (n = 18) or healthy arteries (n = 6),
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Author:Raitoharju, Emma; Oksala, Niku; Lehtimaki, Terho
Publication:Clinical Chemistry
Article Type:Report
Date:Dec 1, 2013
Words:9312
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