MicroRNAs and vascular (dys)function

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs, that control diverse cellular functions by either promoting degradation or inhibition of target messenger RNA translation. An aberrant expression profile of miRNAs has been linked to human diseases, including cardiovascular dysfunction. This review summarizes the latest insights in the identification of vascular-specific miRNAs and their targets, as well as their roles and mechanisms in the vasculature. Furthermore, we discuss how manipulation of these miRNAs could represent a novel therapeutic approach in the treatment of vascular dysfunction.

Introduction

MircoRNAs (miRNAs) are an evolutionarily conserved family of short non-coding RNAs, 18–24 nucleotides in length, that regulate the expression of protein-coding genes. (Wu and Belasco, 2008, Zhao and Srivastava, 2007). It is estimated that the human genome encodes more than 1000 miRNA genes (Bentwich et al., 2005, Berezikov et al., 2005), which regulate the activity of ~ 50% of the genome (Filipowicz et al., 2008). MiRNAs are initially transcribed by RNA polymerase II in the nucleus to form long primary transcripts (pri-miRNA) (Kim, 2005). The pri-miRNAs are processed by the RNase III enzyme Drosha (Lee et al., 2003), to generate shorter stem-loop precursors (pre-miRNA) which can then be exported by the nuclear export factor exportin-5 in a RAN-GTP-dependent manner (Bohnsack et al., 2004, Kim, 2005, Yi et al., 2003). In the cytoplasm, pre-miRNAs are processed further by the RNase III enzyme Dicer into mature 20- to 24-nt miRNAs (Bartel, 2004), which can then be incorporated into the RNA-induced silencing complex (RISC) (Hammond et al., 2001). Regulation of both at the Drosha- and Dicer-processing level is rather complex and details within the cardiovascular system have been recently summarized (Bauersach and Thum, 2011). The RISC complex directs the miRNA to the 3′UTR of the target mRNAs and translationally repress or cleave the mRNA depending on the degree of complementarity between the miRNA and its target (Bartel, 2004, Miranda et al., 2006, van Rooij et al., 2007).

MiRNAs are involved in the regulation of diverse cellular processes, such as proliferation, differentiation, cellular migration and apoptosis (Karp and Ambros, 2005, Miska et al., 2004, Xu et al., 2004, Fleissner et al., 2010). MiRNAs function to ‘fine-tune’ the expression of genes that are important for development and tissue homeostasis (Liu and Olson, 2010). Under cell stress conditions deregulation of miRNAs is often observed and may result in the development of disease, including cardiovascular disorders (such as heart failure (Ikeda et al., 2007, Matkovich et al., 2009, Thum et al., 2007, van Rooij et al., 2006, Thum et al., 2008), cardiac hypertrophy, and post-myocardial infarction remodeling (Roy et al., 2009, van Rooij et al., 2008)).

In vascular cells, especially endothelial and smooth muscle cells, the action of specific miRNAs is important for vascular signaling and function. Some endothelial-specific miRNAs play important roles in angiogenesis, the process by which new blood vessels form through the growth of existing blood vessels in response to environmental cues (Carmeliet, 2005). The involvement of miRNAs in the regulation of angiogenesis was shown by Dicer knockout mice, that died early during development due to impaired angiogenesis (Kuehbacher et al., 2007, Yang et al., 2005). Hence, many studies aimed in studying the role of individual miRNAs in endothelial cells and their importance for angiogenesis. For example, investigators identified miR-126 as a pro-angiogenic miRNA and the clustered miRNAs miR-221 and miR-222 as anti-angiogenic miRNAs (Urbich et al., 2008). Additionally, these miRNAs together with others are often found to be deregulated in vascular disease, such as atherosclerosis, coronary artery disease, post-angioplasty restenosis and diabetic vascular complication (Callis et al., 2007, Hammond, 2006, Mann, 2007, Wiemer, 2007, Yang and Wu, 2007). Recently also reduced levels of circulating miR-126 in the blood of patients with coronary artery disease and diabetes have been described (Fichtlscherer et al., 2010, Zampetaki et al., 2010).

In the following we overview the current knowledge about individual miRNAs for vascular (dys)function (Table 1).

A major component of the blood–brain barrier are cerebral vascular endothelial cells which maintain cerebral homeostasis under physiological conditions. This blood–brain barrier is disrupted by ischemia-induced cerebral injury due to degeneration of cerebral vascular endothelial cells whereas an increase of vascular permeability leads to brain damage and postischemic secondary injury (Sandoval and Witt, 2008). A member of the peroxisome proliferator-activated receptors (PPARs) is PPARδ that has a potential neuro-protective function in ischemic stroke (Bordet et al., 2006). PPARδ is mainly expressed in the vasculature and brain (Chen et al., 2003, Hamblin et al., 2009), and plays a significant role in vascular remodeling, angiogenesis and both vascular and neuronal protection. It has been observed that after oxygen-glucose deprivation in cerebral vascular endothelial cells, an ischemia-like insult, the PPARδ expression level decreases, leading to induction of cerebral vascular endothelial cell apoptosis and necrotic cell death (Yin et al., 2010). In contrast pharmacological activation of PPARδ by intracerebroventricular infusion of the PPARδ agonist GW 501516 (2-[2-methyl-4-[[4-methyl-2-[4-trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid) significantly decreases cerebral endothelial degeneration and improves blood–brain barrier permeability after transient focal cerebral ischemia in mice (Yin et al., 2010). After activation PPARδ binds to a putative peroxisome proliferator hormone response element (PPRE) site in the miR-15a promoter region, which in turn leads to the repression of miR-15a translation (Yin et al., 2010). A downstream target of miR-15a is the antiapoptotic protein bcl-2, whose translation is inhibited by direct binding of miR-15a to the 3′UTR of bcl-2 mRNA (Cimmino et al., 2005). The transcriptional repression of miR-15a by PPARδ increases bcl-2 protein level, reduces caspase-3 activity and subsequent fragmentation of the Golgi apparatus, which in turn leads to decreased blood–brain barrier disruption and protection of the cerebral vascular endothelial cells from apoptosis (Yin et al., 2010) (see Fig. 1). Thus, the cerebrovascular protective role of PPARδ in stroke models could be used as potential therapeutic tool by either pharmacological activation of PPARδ or inhibition of its target miR-15a.

One of the best investigated polycistronic miRNA clusters is miR-17-92, which is located in the intron 3 of the Chromosome 13 open reading frame 25 in the human genome (Ota et al., 2004), and encodes seven miRNAs: miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92-1. Additionally, the transcript contains four exons, that encode the miRNA host gene 1 (MIRHG1), whose function is so far unclear. The main functions of miR-17-92 include modulation of cell proliferation and apoptosis (O'Donnell et al., 2005, Pickering et al., 2009), and many members are also well known for their oncogenic functions (He et al., 2005), such as tumor angiogenesis and increased development of lymphomas. It has been reported, that miR-17-92 members in synergism with the proto-oncogene c-Myc, are upregulated in hematopoietic tumor cells and other solid tumors, where they augment angiogenesis by a paracrine mechanism (Dews et al., 2006). For instance, tumor cells suppress the release of soluble anti-angiogenic factors, like thrombospondin-1 (TSP1), connective tissue growth factor (CTGF) and clusterin, which are targeted by miR-18a, miR-19a, and c-Myc, respectively (Dews et al., 2006, Dews et al., 2010) (see Fig. 1). The presence of c-Myc binding sites in the miR-17-92 promoter region (O'Donnell et al., 2005) permits a miR-17-92 upregulation in c-Myc overexpressing tumor cells. In proliferative cells, c-Myc, along with its interactor Miz1, is able to induce cell cycle progression via binding to promoters of cyclin-dependent kinase inhibitors (CDKI), such as p21CIP1 and m15Ink4b (Warner et al., 1999). Upon activation of the TGFβ signaling pathway, Smad complexes occupy cyclin-dependent kinase inhibitor gene promoters, stimulate cyclin-dependent kinase inhibitor expression, block entry into the cell cycle and lead to c-Myc downregulation. During this competition for binding to promoters of cyclin-dependent kinase inhibitors, c-Myc succeeds by an indirect mechanism, in which miR-17-92 blunt the TGFβ pathway by directly targeting the type II TFGβ receptor and key effector Smad4 (Dews et al., 2010) (see Fig. 1). Hence, c-Myc contributes to an angiogenic phenotype in tumor cells, in part by increasing miR-17-92 levels and blunting TGFβ signaling, which leads to downregulation of antiangiogenic factors. The cluster members miR-17 and miR-20a significantly increase cell-cycle progression by downregulation of the proapoptotic protein bcl-2-interacting mediator of cell death (BIM) (Fontana et al., 2008) and targeting transcription factors of the E2F family (O'Donnell et al., 2005, Pickering et al., 2009), which are important cell cycle regulators. MiR17/20a perform an oncogenic capacity by downregulation of the cell cycle inhibitor and tumor suppressor protein p21, which stimulates proliferation and reduces migration (Doebele et al., 2010, Fontana et al., 2008, Inomata et al., 2009). A repressor of the angiogenic program in endothelial cells is miR-92a, which decreases the expression of proangiogenic proteins like the cell adhesion molecule integrin subunit α5 and αv, histone deacetylase SIRT1 and sphingosine-1-phosphate receptor 1 (SIP1) (Bonauer et al., 2009). Thus, the proangiogenic activity of the miR-17-92 cluster is not related to all members of the miR-17-92 cluster and none of the members is able to induce sprout formation in endothelial cells, as demonstrated by overexpression of every single member of the miR-17-92 cluster in tumor cells, which significantly inhibited three-dimensional spheroid sprouting in vitro (Doebele et al., 2010). Inhibition of miR-17 and -20a using antagomirs reduced tumor growth, increased the number of perfused vessels in matrigel plugs, and enhanced angiogenic sprouting in vivo (Doebele et al., 2010). A systemic administration of miR-92a antagomirs after myocardial infarction and ischemia in murine models led to functional recovery of damaged tissue by augmented neovascularization, increased sprout formation and recovery of blood flow (Bonauer et al., 2009). These observations provide an interesting therapeutic strategy to enhance angiogenesis and reduce tumor growth by use of antagomirs designed to modulate specific members of the miR-17-92 cluster.

In the vasculature endothelial cells are directly exposed to mechanical forces created by pulsatile blood pressure and flow. This mechanical force is essential for regulating vascular signaling and gene expression in endothelial cells, including expression of miRNAs, for modulation of cell morphology, migration, growth, proliferation, apoptosis, production of vasoactive substances (Chien, 2006, Jazbutyte and Thum, 2010). It has been observed that in straight sections of the vasculature, where unidirectional shear stress is present, the expression of different miRNAs is reinforced, including miR-21 (Weber et al., 2010). This miRNA is abundantly expressed in all types of cardiovascular cells, such as vascular smooth muscle cells (VSMCs) (Ji et al., 2007), endothelial cells (Suarez et al., 2007), cardiomyocytes (Cheng et al., 2007), cardiac fibroblasts (Roy et al., 2009), and angiogenic progenitor cells (Fleissner et al., 2010), although its expression is enriched in fibroblasts (Thum et al., 2008). Furthermore, the miR-21 gene is located on chromosome 17q23.2 within the protein-coding region of the transmembrane protein 49 (TMEM49) and is transcribed independently from its own intronic promoter (Fujita et al., 2008). The importance of miR-21 regulation has been recently reviewed (Regalla et al., 2011). The expression pattern of miR-21 changes under hypoxic conditions, as observed in human pulmonary artery smooth muscle cells, where miR-21 was 3-fold upregulated after 6 h of hypoxia (Sarkar et al., 2010). Next, the endogenous nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) activates miR-21 expression in human angiogenic progenitor cells, which leads to increased oxidative stress and reduced the NO bioavailability in these cells due to modulation of endothelial NO synthase activity (Fleissner et al., 2010). Reduced NO production was only observed in connection with elevated ADMA concentration in the plasma which are found in patients with vascular dysfunction like coronary artery disease (Boger et al., 1998). MiR-21 upregulation is accompanied by suppression of sprouty-2 (SPRY2), phosphatase and tensin homology deleted from chromosome 10 (PTEN) (Ji et al., 2007), programmed cell death 4 (PDCD4) (Lin et al., 2009), and peroxisome proliferator-activated receptor α (PPARα) (Sarkar et al., 2010), all of which are functional target genes of miR-21 (see Fig. 1). The repression of the miR-21 target sprouty-2 induced the ERK–MAP kinase-dependent formation of reactive oxygen species (ROS) and led to angiogenic progenitor cell dysfunction (Thum et al., 2008). A strong repression upon miR-21 overexpression in angiogenic progenitors has also been observed for superoxide dismutase 2 (SOD2) (Fleissner et al., 2010). As SOD2 plays a major role in oxidative stress defense its downregulation leads to increased oxidative stress (see Fig. 1). PTEN normally impairs endothelial NO synthase phosphorylation and NO production, as well as regulates apoptosis via its target molecules phosphoinositol 3-kinase (PI3K) and Akt (Church et al., 2010), hence its downregulation by miR-21 may lead to reduced cellular apoptosis (Weber et al., 2010). In summary, under normal conditions some signaling pathways affected by miR-21 overexpression seem to increase NO production whereas some other may lead to a decrease in NO. To date there is no clear evidence about the in vivo net effect of miR-21 manipulation on NO bioavailability. Oxidative stress in endothelial cells, produced by ROS, leads to activation of specific signaling pathways and expression of redox-sensitive genes that are implicated in cardiovascular functions, like smooth muscle cell growth and induction of an inflammatory response (Nickenig et al., 2002) but the effect is dose-dependent. Very high ROS production generally impairs endothelial function and leads to vascular disease (Kunsch et al., 2004).

A posttranscriptional downregulation of PPARα by miR-21 has a positive impact on cell proliferation and migration, mainly via restoration of cyclin-dependent kinase 2 phosphorylation (Zahradka et al., 2006) and decreasing the cyclin-dependent kinase inhibitor p16^INK4a (Gizard et al., 2005). MiR-21 was classified as a miRNA with oncogenic activity, since it was highly enriched in different tumor samples. This could be explained by downregulation of the tumor suppressor gene programmed cell death protein 4 (Lankat-Buttgereit and Goke, 2009) that normally regulates apoptosis of VSMCs and cardiac cells (Cheng et al., 2009b, Lin et al., 2009). Investigators found that miR-21 is aberrantly expressed in many cardiovascular diseases, including cardiac hypertrophy, cardiac fibrosis (Thum et al., 2008), acute myocardial infarction (Dong et al., 2009), and coronary artery disease (Fleissner et al., 2010). Some pathological vascular smooth muscle cell conditions, such as atherosclerosis and hypertensive disease are related to the production of ROS, which lead to VSMC apoptosis and death. A current study identified, that the treatment of VSMCs with hydrogen peroxide increases miR-21 expression in a dose-dependent manner and causes an anti-apoptotic effect on the cells (Lin et al., 2009). These findings suggest, that miR-21 modulation could be a therapeutic option to improve the function of diverse vascular cells.

Vascular endothelial cells are constantly subjected to hemodynamic forces. In straight sections of the arterial tree the blood flow is steady laminar compared to bifurcations, where the blood flow is disrupted and is often connected to the formation of atherosclerotic lesions (Chien, 2006). Exposure of endothelial cells to pulsatile shear flow leads to modulation of endothelial cell signaling, gene expression and physiological functions (Chien, 2006), and to a different miRNA expression profile (Wang et al., 2010). In straight sections of the arteries the endothelial cells respond to pulsatile shear with anti-proliferative and anti-inflammatory effects (Wang et al., 2006), in contrast to bifurcations, where the opposite effects were observed (Hsiai et al., 2003, Matharu et al., 2006). MiRNA expression profiling revealed 21 differentially expressed miRNA 24 h after pulsatile shear stress exposure, including upregulation of miR-23b and of its partner miR-27b (Wang et al., 2010), both of which are clustered together within 0.5 kb of each other on human chromosome 9. MiR-23b and miR-27b are upregulated under pulsatile shear as compared to static conditions (Wang et al., 2010). Upregulation of miR-23b decreases the expression level of the transcription factor E2F1, and phosphorylation of the tumor suppressor protein retinoblastoma (Rb) (Wang et al., 2010). The hypophosphorylated retinoblastoma binds E2F family proteins to suppress endothelial cell growth (Giacinti and Giordano, 2006, Sun et al., 2007) (see Fig. 1). A recent study revealed, that miR-23 and miR-27 have pro-angiogenic function via their target molecules Sprouty2 and Sema6A (Zhou et al., 2011). Repression of these miRNAs by locked nucleic acid-modified anti-miRNAs inhibits angiogenesis in vivo as well as postnatal retinal vascular development in vivo (Zhou et al., 2011). The knowledge of potential miRNAs in mechanotransduction is important for discovery of the regulation of cardiovascular homeostasis under physiological and pathological conditions.

The miR-24 gene is located at two genomic loci within miRNA clusters. The catabolizing-9 encodes the miR-24-1 gene cluster, which includes miR-23b, miR-27b and miR-24-1, and the miR-24-2 gene cluster, which is located on human chromosome 19, encoding miR-24-2, miR-27a and miR-23a. The three miRNAs of the latter cluster are regulated by platelet-derived growth factor subunit B (Chan et al., 2010), a potent inducer of the synthetic, proliferative phenotype. The induction of miR-24 by platelet-derived growth factor subunit B leads to downregulation of its target Tribbles-like protein-3 (Chan et al., 2010), which in turn mediates the degradation of Smad ubiquitin-regulatory factor-1 (Smurf1) proteins (Chan et al., 2010). These proteins generate an antagonistic action of the transforming growth factor (TGF-β) or bone morphogenetic protein (BMP) signaling pathway via degradation of Smad proteins and RhoA (Wang et al., 2007a, Wang et al., 2007b) that are both critical mediators of the contractile phenotype in VSMCs (Chan et al., 2010, Lagna et al., 2007) (see Fig. 1). An antagonism between miR-24 and the TGF-β/BMP signaling pathway has also been observed during erythropoiesis, whereas miR-24 inhibits activin-dependent erythropoiesis by targeting activin type-I receptor gene (Wang et al., 2008a). Thus, miR-24 was investigated as a key regulator of the crosstalk between the pro-contractile TGF-β and BMP signal and the pro-synthetic PDGF signal (Chan et al., 2010). An aberrant miR-24 expression has been observed after hypoxia, and in various tumors, including pancreatic adenocarcinomas (Lee et al., 2007) and uterine leiomyomas (Wang et al., 2007a, Wang et al., 2007b), and indeed miR-24 is enriched in endothelial cells (Zhou et al., 2011) with additional roles in angiogenesis. By targeting the transcription factor GATA2 and p21-activated kinase PAK4, miR-24 prevents endothelial capillary network formation on matrigel and represses endothelial cell sprouting. It has been recently observed, that a pharmacologic miR-24 knockdown by antagomirs augmented vascularity and repressed endothelial apoptosis leading to restriction of myocardial infarct size (Fiedler et al., 2011).

VSMCs have the ability to modulate their phenotype from a quiescent, contractile state to a proliferative, synthetic state in response to many stimuli (Owens, 1995). An aberrant VSMC plasticity has been identified in a variety of vascular disorders, such as post-angioplasty restenosis, atherosclerosis, or abdominal aortic aneurysm formation (Cai, 2006, Doran et al., 2008, Inoue and Node, 2009, Thompson et al., 1997). An important regulator of VSMC physiology is miR-26a that is significantly upregulated during smooth muscle cell differentiation. This highly conserved miRNA increases vascular SMC proliferation while inhibiting cellular differentiation and apoptosis in part via its targeting of downstream elements of the TGF-β/BMP superfamily of growth factors. Among those targets are SMAD-1 that primarily belongs to the BMP-responsive pathway and Smad4, an element of the common TGF-β and BMP pathway, whose action is inhibited by miR-26a (Leeper et al., 2011) (see Fig. 1). MiR-26a plays an important role in controlling phenotype shifting and maintenance of the balance of cells in synthetic, proliferative and contractile, quiescent states (Leeper et al., 2011). Additionally, miR-26a promotes smooth muscle cell proliferation and migration, while inhibiting apoptosis and cell differentiation. Hence, modulation of this miRNA may have a therapeutic effect on inhibition of the progress of many vascular disorders, such as abdominal aortic aneurysm formation, in which aberrant smooth muscle cell plasticity has been observed.

A potent vasoconstrictive peptide and mitogen is endothelin-1 (ET-1) (Yanagisawa et al., 1988). Under normal physiologic conditions vascular endothelial cells release very low levels of the ET-1 peptide (Frelin and Guedin, 1994). The ET-1 expression level increases, however, in response to a number of different stressors, including oxidized low-density lipoprotein (oxLDL) and overall vascular endothelial injury (Boulanger et al., 1992, Xu et al., 2008). The synthesis of ET-1 starts with the expression of the precursor protein preproET-1, which is processed by a prohormone convertase and proteolytic cleaved by endothelin-converting enzyme-1 (ECE-1) to generate ET-1 (Rubanyi and Polokoff, 1994). Elevated levels of ET-1 have been implicated in many pathophysiological processes including atherosclerosis, cardiac hypertrophy, hypertension and cancer progression (Barton et al., 1998, Grant et al., 2003, Hocher et al., 1999, Ito et al., 1993). Recently identified negative regulators of ET-1 transcription are miR-125a-5p and miR-125b-5p, which are highly expressed in vascular endothelial cells, aorta, brain and lung (Li et al., 2010). Both miRNAs belong to the miR-125 family, share an identical ‘seed sequence’, but their genomic locations are different. Therefore, their expression may be regulated by different promoters and transcription factors. In the endothelium miR-125a-5p and miR-125b-5p inhibit the expression of ET-1 by directly targeting two binding sites in the 3′UTR of preproET-1 mRNA (Li et al., 2010) (see Fig. 1). It has been demonstrated that miR-125a-5p and miR-125b-5p in vascular endothelial cells are regulated by oxLDLs. The expression level of miR-125a-5p initially increases more than four-fold after 6 h stimulation with oxLDL, whereas that of miR-125b-5p is constitutively decreased (Li et al., 2010). These opposing effects of oxLDL on miR-125a-5p and miR-125b-5p expression may adjust the transcription of oxLDL-mediated ET-1 production (Li et al., 2010). Experiments with stroke-prone spontaneously hypertensive rats revealed that miR-125a-5p and miR-125a-5p were downregulated in the blood vessels compared with normotensive Wistar–Kyoto rats, whereas the target preproET-1 was upregulated (Jesmin et al., 2006, Sharifi et al., 1998, Stasch et al., 1995). These findings demonstrate that the endothelial miRNAs miR-125a-5p and miR-125b-5p have a considerable impact in vasomotor homeostasis and might serve as interesting therapeutic targets.

MiR-126 is the only miRNA suggested to be specifically expressed in endothelial cells and hematopoietic progenitor cells (Fitch et al., 2004, Kuehbacher et al., 2007, Landgraf et al., 2007, Wang et al., 2008c). The expression pattern of miR-126 in highly vascularized tissues parallels that of epidermal growth factor-like domain 7 (Egfl7) gene (Fitch et al., 2004, Soncin et al., 2003) in which miR-126 (miR-126-3p) and its complement miR-126* (miR-126-5p or miR-123) are encoded within (Fish et al., 2008, Wang et al., 2008c). Both miRNAs derive from the same pri-miRNA (Soncin et al., 2003) and are highly conserved from lower species to Homo sapiens (http//microrna.sanger.ac.uk/sequences/index.shtml). The Egfl7 gene encodes the endothelial cell-restricted EGFL7 protein also known as VE-statin, MEGF7, Notch4-like protein or Zneu1, that act as a chemoattractant and inhibitor of smooth muscle cell migration (Campagnolo et al., 2005, Fitch et al., 2004, Parker et al., 2004, Soncin et al., 2003). The transcription of Egfl7/miR-126 is regulated by two members of the E26 transformation-specific sequence factor (Ets) family namely Ets-1 and Ets-2 which interact with an Ets binding element in a genomic region upstream of the Egfl7/miR-126 gene in order to induce their expression (Harris et al., 2010). Many investigators have focussed on miR-126 function in endothelial cells and revealed that it has a potential function on vascular development and angiogenesis in vivo (Fish et al., 2008, Kuhnert et al., 2008, Wang et al., 2008c) as well as a role in cardiovascular diseases and cancer formation (Musiyenko et al., 2008, Saito et al., 2009, Sun et al., 2010, van Solingen et al., 2009). It has been observed that miR-126 promotes its pro-angiogenic action in part through suppression of Sprouty-related EVH1 domain-containing protein-1 (Spred-1), that normally inhibits the mitogen-activated protein (MAP) kinase pathway by binding and inactivation of rapidly growing fibrosarcoma (Raf), an upstream target of MAP kinase pathway (Nonami et al., 2004, Taniguchi et al., 2007, Wakioka et al., 2001) (see Fig. 1). Spred-1 is also implicated in inhibition of Rho-mediated actin reorganization. Consequently Spred-1 functions as an inhibitor of cell proliferation and migration in response to growth factor signaling (Wakioka et al., 2001). Another mechanism as to how miR-126 achieves its pro-angiogenic function is by decreasing the expression of phosphatidyl-inositol 3-kinase regulatory subunit beta (PIK3R2), which inhibits the activity of phosphatidyl-inositol 3-kinase (Ueki et al., 2003) (see Fig. 1). Besides the potential role in angiogenesis, miR-126 is also involved in vascular inflammation by suppression of vascular cell adhesion molecule-1 (VCAM-1) (Harris et al., 2008). Resting cells normally show low expression of adhesion molecules whereas the presence of cytokines induces adhesion molecule expression and mediates leukocyte adherence to endothelial cells (Salmi and Jalkanen, 2005, Weber, 2003). Downregulation of VCAM-1 expression by miR-126 may function to suppress inflammation in resting cells, by diminished leukocyte interactions with endothelial cells and infiltration of leukocytes into the vascular wall (van Rooij et al., 2007). Other repressed miR-126 target molecules are the serine/threonine kinase p21-activated protein kinase (pak1) which activity is required for endothelial motility (Kiosses et al., 1999) and permeability (Stockton et al., 2004), EphrinB2, an inhibitor of the MAP kinase signaling downstream of VEGF (Kim et al., 2002), as well as regulator of G-protein signaling 5 (RGS5), which decreases phosphorylation of ERK (Cho et al., 2003) and regulator of G-protein signaling 4 (RGS4), a related RGS protein that negatively regulates tubulogenesis by reducing ERK phosphorylation (Albig and Schiemann, 2005, Liu et al., 2009a) (see Fig. 1). Mentionable is the function of miR-126 as tumor suppressor gene in lung cancer cells and the observation that this endothelial enriched miRNA is downregulated in many lung cancer cell lines (Matkovich et al., 2009). The investigation of an inhibitory effect of miR-126 on VEGF expression by targeting a binding site in its mRNA 3′UTR, which is normally upregulated during both angiogenesis and tumor expansion, suggests the opportunity that delivery of miR-126 could be a therapeutic intervention in human lung cancer treatment (Liu et al., 2009a). Next to tumorigenesis, miR-126 is also required after myocardial infarction when injured vessels at the side of the infarct need to develop collateral vessels in the ischemic myocardium (Semenza, 2003). Hence strategies to increase miR-126 levels may be a beneficial effect in case of pathological vascularization. Recently, there has been growing interest in circulating miRNAs (Gupta et al., 2010, Mitchell et al., 2008), which correlate with the onset of myocardial injury, coronary artery disease and heart failure (Fichtlscherer et al., 2010, Ji et al., 2009, Tijsen et al., 2010, Wang et al., 2008c). It has been observed that miR-126 is reduced in plasma of patients with type 2 diabetes mellitus. The miR-126 content of endothelial apoptotic bodies is also reduced after the presence of high glucose concentrations (Zampetaki et al., 2010). The loss of miR-126 in association with type 2 diabetes mellitus suggests miR-126 as a novel biomarker for cardiovascular risk evaluation and classification (Zampetaki et al., 2010).

A spontaneous switch of quiescent endothelial cells to a more proliferative state is a rare event (Hobson and Denekamp, 1984) unless the presence of angiogenic growth factors including VEGF and basic fibroblast growth factor (bFGF). This angiogenic switch is a feature during pathological neovascularization, especially tumorigenesis (Folkman, 1992). There is evidence that miRNAs also participate in the activation of quiescent endothelium. Here, miR-132 has been described to act as an angiogenic switch to induce neovascularization (Anand et al., 2010). A high expression level of miR-132 was detected in activated endothelial cell during both developmental and pathological neovascularization as well as in the endothelium of human tumors and hemangiomas (Anand et al., 2010). In contrast miR-132 is undetectable in normal endothelium suggesting miR-132 to serve as a marker of activated endothelium (Anand et al., 2010). The transcription of miR-132 from an intergenic region on human chromosome 7 is regulated by the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) (Nudelman et al., 2010, Vo et al., 2005) which can be rapidly induced by VEGF and bFGF (Mayo et al., 2001, Tan et al., 1996). An upregulated expression of miR-132 in activated endothelial cells results in considerably increased cell proliferation and tube formation by downregulation of p120RasGAP (Anand et al., 2010). Administration of anti-miR-132 blocked growth factor-induced angiogenesis in vitro and in vivo by maintaining vessels in the resting state (Anand et al., 2010). The target molecule p120RasGAP encoded in the Rasa1 gene is expressed in normal but not in tumor endothelial cells and functions as a negative regulator of vascular development and remodeling (Henkemeyer et al., 1995) via suppression of the proto-oncogene Ras (Anand et al., 2010). Thus, suppression of p120RasGAP by miR-132 in the activated endothelium increases Ras activity and results in pathological neovascularization (Anand et al., 2010) (see Fig. 1). These findings could be useful for novel antiangiogenic therapy in a wide range of pathological conditions by the delivery of miR-132 antagomirs since current therapies, which target single pathways such as the VEGF pathway, can cause tumor resistance by upregulation of alternative growth factors.

The nonhomologous clustered miRNAs miR-143 and miR-145, which originate from the same transcriptional unit, are well known for their crucial role in SMC differentiation and vascular pathogenesis (Elia et al., 2009). These miRNAs are most abundant in vessels and SMC-containing organs, both during development and in late fetal and postnatal stages, where the expression level is decreasing in the heart, but remain constitutively expressed in smooth muscle cells (Elia et al., 2009). Additionally, miR-145 is moderately expressed in fibroblasts (Zhang, 2009) and a strong expression level of miR-143 was detected in adipocytes (Esau et al., 2004) and osteoclasts (Palmieri et al., 2008). MiR-145, but not miR-143, is able to direct VSMC differentiation from embryonic stem cells (Cordes et al., 2009). In VSMCs, miR-145-mediated phenotypic modulation from a contractile, quiescent phenotype to a synthetic, proliferative phenotype occurred by repression of its target genes krüppel-like factor 4 (KLF4), a transcription factor involved in pluripotency (Takahashi et al., 2007) and krüppel-like factor 5 (KLF5), which in turn increases the expression of its downstream signal molecule myocardin (Cheng et al., 2009a). This potent co-activator interacts with serum response factor (SRF) to activate most, but not all, genes associated with contractile smooth muscle cell phenotype (Chen et al., 2006) (see Fig. 1). The activation of cytoskeletal protein genes is also induced by myocardin-related transcription factors, which are released from monomeric G-actin in the cytoplasm, to translocate to the nucleus, where they bind to SRF to drive gene expression of smooth muscle cell contractile proteins (Kuwahara et al., 2005, Medjkane et al., 2009, Miralles et al., 2003). In human embryonic stem cells, miR-145 targets octamer binding transcription factor (OCT4), and SRY-box 2 (SOX2) and krüppel-like factor 4, which are important transcription factors in induced pluripotent stem cells (Xu et al., 2009). It has been reported, that miR-145 selectively targets c-Myc, rhotekin, insulin receptor substrate-1 and β-actin in cancer cells, and have inhibitory effects on cancer cell growth (Sachdeva et al., 2009, Takagi et al., 2009, Wang et al., 2009a). There are many other miR-145 targets that are involved in actin dynamics and cytoskeletal function, like facin (Quintavalle et al., 2010), actin, cofilin, calmodulin kinase IIδ (CamkIIδ), adducin-3 (Add3) (Barkalow et al., 2003, Gardner and Bennett, 1987), sling-shot 1 (Ssh1) (Eiseler et al., 2009) and sling-shot 2 (Ssh2) (San Martin et al., 2008). Unlike miR-145, miR-143 targets Elk-1, a member of the E-twenty six (ETS) oncogene family, that acts as a myogenic repressor and an activator of VSMC proliferation by displacing myocardin from SRF (Cordes et al., 2009). Further miR-143 target genes include protein kinase C-ε and PDGF receptor α (Quintavalle et al., 2010), which participate in cell migration and proliferation (Heldin and Westermark, 1999). Accordingly, miR-143 and miR-145 cooperatively target many connected transcription factors to promote differentiation and simultaneously repress proliferation of VSMCs by the coincidence of SRF-dependent co-activators and co-repressors. It has been observed, that downregulation of miR-143/145 by PDGF is a key step in the formation of podosomes (Quintavalle et al., 2010), which are dynamic, short-lived, actin-rich protrusions of the plasma membrane and a morphological feature of migrating VSMCs (Gimona et al., 2003, Linder and Aepfelbacher, 2003). The expression level of miR-143/145 is downregulated in some vascular diseases, including injured or atherosclerotic vessels (Cordes et al., 2009) and in diverse cancer cell lines (Calin and Croce, 2006). It is known, that miR-145 has a strong inhibitory effect on cancer cell proliferation and hence is described as a novel tumor suppressor gene (Cho et al., 2009, Liu et al., 2009c). In various cancer cell lines, like colon cancer (Michael et al., 2003, Schepeler et al., 2008) and breast cancer (Iorio et al., 2005, Sempere et al., 2007), miR-145 was distinguished as one of the most downregulated miRNAs. In summary, miR-145 may have important consequences for the diagnosis and therapy of proliferative cardiovascular disease.

Another miRNA that participates in the phenotypic control of VSMCs is miR-146a. This miRNA promotes VSMC proliferation and neointimal hyperplasia in vivo (Sun et al., 2011). It also plays an important role in innate immune responses and cancer metastasis (Williams et al., 2008). It has been reported that miR-146a was upregulated in balloon-injured carotid arteries of rats (Ji et al., 2007). In contrast, transfection of antisense miR-146a oligonucleotides considerably decreased neointimal hyperplasia in these arteries (Sun et al., 2011). This effect could be associated with the miR-146a target gene krüppel-like factor 4, which has an antiproliferative effect on VSMCs by upregulating the potent cyclin-dependent kinase inhibitor p21 (Sun et al., 2011, Wassmann et al., 2007) and two VSMC differentiation genes smooth muscle-22α and α-smooth muscle actin, and also by downregulation of the embryonic smooth muscle myosin heavy chain gene (Wang et al., 2008a, Wang et al., 2008b, Wang et al., 2008c, Wang et al., 2008d). MiR-146a directly targets the 3′UTR of krüppel-like factor 4 (KLF4) and represses its transcription (Sun et al., 2011) (see Fig. 1). In turn, krüppel-like factor 4 inhibits miR-146a transcription by binding a CACCC (or GGGTG) sequence in the miR-146a promoter region, thus both miR-146a and krüppel-like factor 4 form a feedback loop to adjust each other's expression (Sun et al., 2011). Another transcription factor of the krüppel-like factor family is krüppel-like factor 5 (KLF5) that competes with krüppel-like factor 4 to regulate the miR-146a promoter via binding to the similar DNA promoter sequence (Sun et al., 2011). Krüppel-like factor 5 exerts an opposing effect on miR-146a transcription (Dang et al., 2002), and hence functions as a positive regulator of cell proliferation (Sun et al., 2011). The finding that miR-146a is a mediator of VSMC proliferation and neointimal hyperplasia may have implications for the development of miRNA-based therapies of proliferative vascular disease.

MiR-210 belongs to the strongest hypoxia-induced miRNAs and has an important function in cell survival and angiogenesis (Ivan et al., 2008). The increased expression of miR-210 in endothelial cells in response to low oxygen tension leads to upregulation of several angiogenic factors, inhibition of caspase activity, and prevention of cell apoptosis (Hu et al., 2010). One important target of miR-210 is ephrin-A3 (Fasanaro et al., 2008) that plays a crucial role in the development of the cardiovascular system and in vascular remodeling by binding to its ephrin receptor (Kuijper et al., 2007). MiR-210 also targets protein tyrosine phosphatase 1b (Ptp1b) (Hu et al., 2010) that acts as a negative regulator in vascular endothelial growth factor signaling in endothelial cells (Nakamura et al., 2008). Ephrin-A3 and protein tyrosine phosphatase 1b are downregulated by miR-210 under hypoxic conditions and hence modulate the angiogenic response to ischemia (Hu et al., 2010) (see Fig. 1). Additionally miR-210 targets genes that could participate in this reaction such as the pro-apoptotic kinase death associated-protein kinase 1 and the connective tissue growth factor, a cysteine-rich protein with important roles in angiogenesis, tissue repair and cancer (Hu et al., 2010). The delivery of miR-210 after myocardial infarction was shown to improve heart function by induction of angiogenesis and inhibition of apoptosis in the heart, leading to the assumption, that miR-210-based therapeutic interventions could have beneficial effects in the treatment of ischemic cardiovascular diseases.

The blood vessel patterning is guided by transmembrane receptors that control cell migration and pathfinding upon molecular signals (Carmeliet and Tessier-Lavigne, 2005). These receptors include roundabout (Robo) receptors and function together with their Slit ligands as important regulators of both axon and vascular guidance (Brose et al., 1999). The influence of the Robo receptor on cell migration can be positive or negative depending on the isoform composition and the cellular context (Legg et al., 2008, Wang et al., 2003). Important mediators for Slit–Robo signaling are heparin sulfate proteoglycans (HSPG) that are transmembrane or secreted proteins covalently linked to heparin sulfate chains (Hussain et al., 2006). It has been reported that these proteins are crucial for the transport of signal molecules through the extracellular matrix, formation of growth factor gradients, and for strengthening of the interaction between ligand and receptor, including VEGF, fibroblast growth factor, and Slit–Robo pathway (Gitay-Goren et al., 1992, Inatani et al., 2003, Johnson et al., 2004, Yayon et al., 1991). The expression of Robo receptors is regulated by miR-218, an evolutionary conserved miRNA, which is encoded within an intron of the Slit genes Slit2 and Slit3 (Tie et al., 2010). High expression level of miR-218 has been observed in brain and eye. In the latter miR-218 has an impact on retinal angiogenesis, and controlling the density of the capillary plexus (Small et al., 2010). MiR-218 directly represses the expression of Robo receptor 1 and Robo receptor 2, thereby generating a negative feedback loop in response to Slit gene activation, which leads to separating ligand from the receptor (Small et al., 2010). Other target genes of miR-218 are components of the heparin sulfate proteoglycans biosynthetic pathway, such as heparin sulfate-modifying enzyme glucuronyl C5-epimerase (GLCE), 2-O and 6-O sulfotransferases, which are all critical enzymes in regulating the heparin sulfate proteoglycan activity (Jia et al., 2009). Consequently the modulation of heparin sulfate proteoglycans by miR-218 may influence the Slit–Robo receptor signaling by different mechanisms leading to reduced endothelial cell migration. It has been recently observed that miR-218 inhibits tumor cell migration and metastasis via transcriptional repression of Robo receptor 1 (Tie et al., 2010) (see Fig. 1).

The miRNAs miR-221 and miR-222 have been shown to be significantly upregulated in circulating angiogenic cells (CACs, formally known as endothelial progenitor cells) of patients with coronary artery disease in comparison to that of non-coronary artery disease patients (Minami et al., 2009). Both miRs are clustered together in close proximity on the Xp11.3 chromosome (Altuvia et al., 2005, Calin et al., 2004) and are expressed in endothelial cells, VSMCs (Liu et al., 2009b), as well as in hematopoietic cells (Felli et al., 2005, Poliseno et al., 2006). Analysis of the promoter structure of the miR-221 gene revealed the existence of an E-box, that can recruit microphthalmia-associated transcription factor as observed in melanocytes and other E-box binding proteins after PDGF activation, to induce miR-221 expression (Ozsolak et al., 2008). The function of miR-221/222 in the cardiovascular system is cell-type dependent via regulation of different target genes. In hematopoietic progenitor cells the expression of both miRNAs is markedly decreased during maturation and differentiation of these cells into erythroid cells, which leads to the assumption that these miRNAs normally function to inhibit erythropoiesis (Felli et al., 2005). Under hyperglycemic conditions, miR-221/222 overexpression in CACs downregulates cell differentiation and mobilization via targeting of c-kit, a receptor for stem cell factor (SCF) (Li et al., 2009). In that case, a lipid lowering therapy with statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors), especially atorvastatin has been shown to reduce miR-221/222 levels in patients with coronary artery disease and in mice after myocardial infarction, accompanied by elevated number and mobilization of CAC (Minami et al., 2009). Thus, intervention of the miR-221/c-kit pathway with atorvastatin could be a useful strategy for the treatment of diabetic vascular disease patients. In VSMCs, miR-221/222-mediated downregulation of c-kit reduces the expression level of the potent smooth muscle cell-specific coactivator myocardin, which in turn inhibits smooth and cardiac muscle cell differentiation by repression of smooth muscle cell-specific contractile gene transcription (Davis et al., 2009) (see Fig. 1). Hence, treatment of VSMCs with anti-miR-221 or anti-miR-222 might represent a potential new strategy on vascular proliferative disorders. MiR-221/222 play an additional role in modulation of smooth muscle cell proliferation by targeting the cell cycle inhibitor p27 (Kip1) (Liu et al., 2009b) (see Fig. 1). This effect on VSMCs is consistent with that on cancer cells, in which high miR-221/222 expression levels promote cell proliferative effects due to downregulation of c-kit in addition to down-regulation of p27 (Kip1) (Fornari et al., 2008, Galardi et al., 2007, le Sage et al., 2007). An effective cancer therapy with the drug imatinib mesylate, which is a 2-phenylaminopyrimidine derivate that functions as a specific inhibitor of a number of tyrosine kinase enzymes, has been described (Buchdunger et al., 1996). This drug is able to inhibit of tyrosine kinase activity of both c-kit and PDGF receptor, which have major effects on decreased proliferation and enhanced apoptosis of malignant cells (Ali and Ali, 2007, Gross et al., 2006). The anti-angiogenic function of the miR-221/222 cluster is obtained by controlling the expression of signal transducer and activator of transcription 5 (STAT5), which normally promote new vessel formation (Dentelli et al., 2010). Furthermore, in endothelial cells miR-221 strongly downregulates ZEB2 (also known as SMAD-1 interacting protein (SIP-1)) (Chen et al., 2010), which usually modulates epithelial–mesenchymal transition in other cell types (Lorenzen et al., 2011a, Vandewalle et al., 2005). Downregulation of ZEB2 upregulates the homeobox gene MEOX2 (also known as GAX), which was suggested to decrease angiogenesis through inhibition of nuclear factor-κB-dependent endothelial cell gene expression (Patel et al., 2005) and to increase the expression of p21^WAF/CIP1 (Chen et al., 2007, Smith et al., 1997), leading to decreased endothelial cell proliferation and maintaining the cells in G0/G1 cell cycle arrest (see Fig. 1). Targeting of ZEB2 might be useful for an antiangiogenic therapy of cancer and other angiogenic disorders.

During hemolysis an amount of free heme is released in the vasculature and can readily enter cell membranes based on its hydrophobic feature whereby assisting a cellular oxidant-mediated killing (Kumar and Bandyopadhyay, 2005). A crucial role in metabolizing the heme level plays the microsomal/mitochondrial enzyme heme oxygenase-1 (HO-1) enzyme, which oxidizes protoheme to biliverdin IXα and carbon monoxide (Sugishima et al., 2000). Different agents, including heme, oxidants and hypoxia augment the transcription of HO-1, which protects the cells and tissues against oxidative stress and ischemia–reperfusion injury (Otterbein et al., 2003, Wagener et al., 2003). Additionally the post-transcriptional regulation of HO-1 is also necessary to metabolize the heme magnitude, in which the two miRNAs miR-377 and miR-217 play a major role (Beckman et al., 2011). The 3′UTR of HO-1 exhibits binding sites for both miR-377 and miR-217, and it has been shown that cells transfected with combination of miR-377 and miR-217 cause a distinct reduction of HO-1 protein expression and enzyme activity in comparison to non-transfected control cells (Beckman et al., 2011) (see Fig. 1). In contrast, this effect failed to appear when cells were transfected with either miR-377 or miR-217 alone (Beckman et al., 2011). A possible interaction mechanism could be that a weak binding of miR-217 to the HO-1 3′UTR may help to potentiate the interaction between miR-377 and HO-1 3′UTR, which ultimately results in reduction of HO-1 enzyme activity (Beckman et al., 2011). However the expression of each miRNA is also controlled by different factors, including hemin, that significantly decreases the expression of miR-217 and also to a lower extent the miR-377 expression (Elmen et al., 2008). There is evidence that both miR-377 and miR-217 are implicated in pathogenesis of diabetic nephropathy in human and murine cell lines (Wang et al., 2008b). MiR-377 represses the translation of superoxide dismutase 1 and 2 (SOD1 and 2) and p21/cdc42/Rac1-activated kinase 1 (PAK1), which increases the fibronectin production (Wang et al., 2008b). However, SOD2 is an important enzyme for decreasing toxic ROS in the mitochondria, and carbon monoxide, produced by HO-1 during hemolysis, elevate levels of SOD2 (see Fig. 1). Next to miR-377, SOD2 is also regulated via miR-21 (Fleissner et al., 2010). Thus, it could be assumed that reduced miR-337 levels implicate an antioxidant effect by upregulation of both HO-1 and SOD2 and offer an interesting therapeutic opportunity to protect cells from oxidative stress and damage. This speculation is strengthened by the investigation, that heme has an impact on miRNA processing, since the cofactor of Drosha DiGeorge Syndrome critical region gene 8 (DGCR8) binds heme to develop homodimerization of DGCR8 (Faller et al., 2007). The generated complex includes one heme molecule per homodimer, and is much active than without heme, consequently strengthen miRNA processing (Faller et al., 2007). In this case DGCR8 function as a heme biosensor to modulate for example the synthesis of heme catabolizing enzymes like HO-1 through processing of crucial miRNA molecules. Indeed, animals with a functional DGCR8 knockout specifically in cardiomyocytes develop cardiac failure (Rao et al., 2009).

The miR-15/107 group of miRNAs plays a role in cell division, metabolism, stress defense, and angiogenesis in vertebrates and has a variety of different members. All vertebrates studied so far express miR-15a, miR-15b, miR-16, miR-103, and miR-107, whereas mammals express miR-195, miR-424, miR-497, miR-503, and miR-646. It has been reported that members of this miRNA family are involved in human disease, like cancers and cardiovascular disease. MiRNAs belonging to the miR-15/107 group share the same seed sequence (AGCAGC) near the 5′end of the mature miRNA as well as many target genes (Finnerty et al., 2010). One of these miRNAs is miR-503 whose expression is upregulated in endothelial cells of diabetes mellitus mimicking high d-glucose culture conditions and ischemia-associated starvation culture condition with low growth factors. Wang et al. have also observed that miR-503 is also upregulated in myocardial microvascular endothelial cells from type 2 diabetic Goto–Kakizaki rats, which show impaired angiogenesis when compared with healthy rats (Wang et al., 2009b). In endothelial cells, augmented miR-503 expression leads to reduced proliferation, migration, and network formation on matrigel. These effects are caused by the downregulation of the miR-503 target genes CCNE1 and cdc25A. Delivery of anti-miR-503 to the ischemic adducer of diabetic mice improved the impaired postischemic angiogenesis and blood flow recovery (Caporali et al., 2011). Thus, modulation of miR-503 expression in diabetic patients provides a possible therapeutic tool.

The biogenesis and function of miRNAs are related to the molecular mechanism of various human diseases. There are miRNAs that have been associated with oxidative stress, inflammation, impaired adipogenesis and insulin signaling, apoptosis and angiogenesis, which may lead to the development of obesity, diabetes mellitus type II, cancer, and hypertension (Bonauer et al., 2010, Heneghan et al., 2010). These pathologies are often closely linked to cardiovascular disorders, like atherosclerosis and angiogenesis. For example, infiltration of macrophages is a common feature of inflammation, angiogenesis and cancer. In response to inflammatory stimuli, tumor-supporting macrophages infiltrate in cancer areas, and increase the production of angiogenesis-related factors (Lin and Karin, 2007). The stimulation of angiogenesis assists the progression of cancer. It has been observed, that miR-21 is one of the miRNAs whose expression level changes during inflammatory conditions. miR-21 acts as a mediator in inflammation-associated carcinogenesis (Loffler et al., 2007). The regulation of miR-21 and role in inflammation has recently been reviewed (Kumarswamy et al., 2011). Some miRNAs are implicated in cholesterol and fatty acid metabolism in the liver. Defects in homeostatic regulation of cholesterol and fatty acids are associated with the development of diabetes mellitus type II and atherosclerotic cardiovascular disease (Moller and Kaufman, 2005). A highly liver-restricted miRNA is miR-122 which indirectly acts to control cholesterol/lipid homeostasis (Esau et al., 2006) and deregulations thus indirectly may also affect the cardiovascular system. Another organ system that, if diseased, is associated to cardiovascular disease is the kidney. Here, it is of interest that a miRNA, miR-210, can be found in significantly altered levels in plasma of patients with kidney diseases and thus might be useful as biomarker (Lorenzen et al., 2011b, Lorenzen et al., 2011).