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Curr Atheroscler Rep (2013) 15:352 DOI 10.1007/s11883-013-0352-6 CLINICAL TRIALS AND THEIR INTERPRETATIONS (J PLUTZKY, SECTION EDITOR) Nuclear Reprogramming and Its Role in Vascular Smooth Muscle Cells Silvio Zaina & Maria del Pilar Valencia-Morales & Fabiola E. Tristán-Flores & Gertrud Lund Published online: 24 July 2013 # Springer Science+Business Media New York 2013 Abstract In general terms, “nuclear reprogramming” refers to a change in gene expression profile that results in a significant switch in cellular phenotype. Nuclear reprogramming was first addressed by pioneering studies of cell differentiation during embryonic development. In recent years, nuclear reprogramming has been studied in great detail in the context of experimentally controlled dedifferentiation and transdifferentiation of mammalian cells for therapeutic purposes. In this review, we present a perspective on nuclear reprogramming in the context of spontaneous, pathophysiological phenotypic switch of vascular cells occurring in the atherosclerotic lesion. In particular, we focus on the current knowledge of epigenetic mechanisms participating in the extraordinary flexibility of the gene expression profile of vascular smooth muscle cells and other cell types participating in atherogenesis. Understanding how epigenetic changes participate in vascular cell plasticity may lead to effective therapies based on the remodelling of the vascular architecture. Keywords Vascular smooth muscle cell . Epigenetics . Atherosclerosis . Nuclear reprogramming This article is part of the Topical Collection on Clinical Trials and Their Interpretations S. Zaina (*) Department of Medical Sciences, Division of Health Sciences, León Campus, University of Guanajuato, 20 de Enero no. 929, 37320 León, Gto., Mexico e-mail: [email protected] M. del Pilar Valencia-Morales : F. E. Tristán-Flores : G. Lund Department of Genetic Engineering, CINVESTAV Campus Guanajuato, Irapuato, Gto., Mexico Introduction Nuclear reprogramming (NR) is defined as a change in gene expression profile, which causes a cell’s phenotype to switch to that of an unrelated cell type [1]. NR research was initiated by experiments in amphibia showing that factors present in oocyte cytoplasm can reset a somatic cell’s genome to reexpress oocyte-specific genes [2]. Because of obvious therapeutic implications, those early experiments have prompted extensive efforts to devise techniques to reprogram somatic cells for the production of pluripotent cells or to achieve direct transdifferentiation. This vast field of research has been reviewed recently with a special focus on the cardiovascular system by Ma et al. [3•]. In this review, after an introductory overview of epigenetic mechanisms, we will focus on their participation in the induction of the vascular smooth muscle cell (VSMC) phenotypic switches as encountered in atherosclerosis. One well-established crucial step in atherogenesis is the phenotypic switch of VSMCs from a contractile, quiescent, fully differentiated cell type to a “synthetic” cell capable of extracellular matrix synthesis, migration and proliferation [4]. Another important atherosclerosis-related phenotypic switch is that from VSMCs to osteoblast-like cells during vascular calcification [5]. Furthermore, it has been shown that VSMCs may transdifferentiate to a macrophage-like proinflammatory phenotype. Remarkably, at least some of these phenotypic switches are reversible. VSMCs can regress to a differentiated phenotype, and even macrophages can undergo reverse migration from the vessel to lymph nodes in advanced, spontaneously regressing atherosclerotic lesions [6•]. The dominant view on vascular cell plasticity is that full differentiation and quiescence is the default status of adult vascular cells until a phenotypic switch is triggered as a response to proinflammatory signals associated with atherogenic risk factors or to experimentally induced mechanical injuries [7]. Yet, this model 352, Page 2 of 9 has been challenged by the recent detection of atherosclerotic lesions in preagricultural populations, suggesting that such phenotypic plasticity may be an intrinsic vascular feature regulated by an age-regulated cellular clock [8]. One important and intensely debated issue is whether resident differentiated VSMCs are the only source, or the source at all, of phenotypically switched cells. The latter may arise from recruited circulating progenitor cells or a resident vascular multipotent population, in analogy with proposed models of cancer initiation [9]. The topic of peripheral blood VSMC progenitor cells is extremely promising and intellectually fascinating, but the experimental evidence is at best controversial. Indeed, on the basis of results in animal models, the existence of circulating VSMC progenitor cells has been challenged [10]. The unresolved issues in the field have been thoroughly reviewed elsewhere and will not be touched on here [11•]. The topic of resident vascular multipotent cells has been relatively little explored and is on the rise. A recent report allegedly demolishes the view that resident, pre-existing differentiated VSMCs are the origin of the dedifferentiated counterparts, as it claims that the latter arise solely from resident multipotent cells [12]. Accordingly, previous findings suggest that resident vascular stem-like cells are activated by turbulent blood flow, a known local factor predisposing to atherosclerotic lesion initiation (recently reviewed by Zhang et al. [13]). One expected feature of an atherosclerotic lesion derived from a tiny population of resident or recruited stem-like cells is clonality. Experiments using X-chromosome inactivation as a marker of clonality have been inconclusive, since normal arteries are composed of patches of highly similar VSMCs [14]. In summary, the jury is still out on the issue of whether vascular progenitor cells are recruited from peripheral blood or are resident. One can only expect that pluripotent cell research will propose stimulating and paradigm-breaking views of vascular and general cell biology. Owing to the high degree of uncertainty in the field, our review takes the simplified view that pre-existing vascular VSMCs undergo phenotypic switches characteristic of the atherosclerotic lesion. Whatever the unresolved issues and detailed mechanisms, cellular phenotype switching is a quintessential epigenetic phenomenon, since it represents the reversible emergence of phenotype diversity in the presence of genetic invariance, as exemplified by twin studies [15]. This is possibly an oversimplified view, as it cannot be ruled out that the frequency of somatic de novo mutations is perhaps higher than previously appreciated and thus may play a significant role in the phenomena described here (see later). Keeping the latter caveat in mind, we will focus on epigenetic transcriptional regulation as an important mechanistic and potentially therapeutic clue for vascular cell plasticity. Curr Atheroscler Rep (2013) 15:352 Epigenetics in Cardiovascular Disease: A General View Epigenetics is the study of nuclear information that is additional to the genetic information contained in a cell’s DNA sequence. At the molecular level, the epigenetic information consists of highly dynamic chemical modifications of cytosine residues of DNA and N-terminal tails of histone proteins. Besides these well-understood epigenetic “marks”, non-coding RNAs (ncRNAs) are emerging as additional epigenetic players. The transcriptional impact of epigenetic marks is dependent both on the nature of the specific mark and on the local sequence context. Epigenetic marks are essential regulators of cellular differentiation and the organism’s development–for example the loss of DNA methylation is embryonically lethal in mice [16]. Epigenetics has become increasingly popular in the cardiovascular field in recent years, for at least two reasons. One is the appreciation that cancer and a growing list of other diseases are associated with aberrant epigenomes—defined as the distribution of epigenetic marks across a cell’s genome [17]. Secondly, a large body of evidence cemented the notion that the epigenome can be shaped by a variety of exogenous stimuli. Crucially, some of the latter fall within the same categories as known cardiovascular risk factors: diet, environmental pollutants, smoking and even subtle factors such as behaviour and stress [18–20]. As a result, a novel mechanistic model of cardiovascular disease emerged, according to which risk factors act by imposing aberrant epigenetic marks that result in pathological transcription profiles [21]. In the following sections, we provide a brief description of the best-characterized epigenetic marks, followed by examples of the connection between specific epigenetic mechanisms and vascular cell plasticity in atherosclerosis. DNA Methylation DNA methylation involves the covalent addition of a methyl group to carbon 5 of deoxycytosine, to yield 5methyldeoxycytosine (5mdC). In mammals, most of the 5mdC is in the 5′ position of CpG dinucleotides. DNA methylation is performed by DNA methyltransferases (DNMTs), of which the best characterized are DNMT1, DNMT3A, and DNMT3B. A loose functional specialization exists among these DNMTs, where DNMT1 is mainly involved in maintaining pre-existing DNA methylation profiles during mitosis, whereas the DNMT3A and DNMT3B impose DNA methylation de novo [22]. DNA demethylation is thought to be performed not by a single enzyme, but rather via direct deamination of 5mdC to thymine, or by oxidation of carbon 5 to produce 5-hydroxymethyldeoxycytosine. The latter can be either further oxidized to formylcytosine and carboxycytosine or directly deaminated to 5hydroxymethyluracil [23]. All these intermediates trigger Curr Atheroscler Rep (2013) 15:352 the base excision repair pathway and replacement with an unmethylated cytosine. Active DNA demethylation can occur in the absence of cell proliferation, for example during monocytic differentiation [24•]. As for transcriptional impact, methylation of promoter sequences is generally a repressive mark. On the other hand, gene body methylation is widespread and has been proposed to have pleiotropic functions such as regulation of RNA splicing, silencing of cryptic promoters and elimination of transcriptional noise [25, 26]. Interestingly, gene bodies are targeted for methylation in response to dietary factors in Apis mellifera [27]. Another function of DNA methylation is silencing of transposable elements [28]. A further important notion is that the probability of a given cytosine residue being methylated can be affected by the surrounding DNA sequence, as shown by the association between SNP genotype and local DNA methylation status [29]. Conversely, 5mdC deamination has been proposed as the source for C–T transitions, the single most abundant type of single-base mutations in eukaryotes (see later). These observations demonstrate the epigenetic information and genetic information are not independent, but are rather complementary and can modify each other. Page 3 of 9, 352 results in trimethylation of histone H3 lysine 27 (H3K27me3) [32]. Non-coding RNAs High-throughout analysis of the transcriptome has revealed a plethora of ncRNAs. These are generally divided into families relating to size and function to dynamically regulate gene expression and silence transposable elements. MicroRNAs (miRNAs) are approximately 22-nt small ncRNAs that promote messenger RNA (mRNA) degeneration and/or inhibit their translation by complementarily binding on the 3′ untranslated region of mRNAs. Likewise, ncRNAs of more than 200 nt, denominated long ncRNAs (lncRNAs), have been implicated in both transcriptional and post-transcriptional gene regulation (reviewed by Rin and Chang [33] and Yoon et al. [34]). Transcriptional mechanisms include the association of ncRNAs with known silencing or activator protein complexes such as PcG and Mediator, respectively [35]. Post-transcriptional mechanisms affected by lncRNAs include pre-mRNA splicing, mRNA turnover, translation, miRNA function and, as recently demonstrated, transcriptional interference [34, 36]. Histone Post-translational Modifications De Novo Somatic Mutations Histones are small proteins tightly bound to DNA. Histones are fundamental components of chromatin, a highly ordered nucleoprotein polymer. The basic unit of chromatin is the nucleosome, which consists of approximately 146 DNA base pairs wrapped around an octamer of two copies of each of the histones H2A, H2B, H3 and H4. Nucleosomes are characteristically absent from gene promoter transcription start sites and generally co-localize with methylated DNA [30]. The Nterminal histone tail can undergo a plethora of posttranslational modifications: acetylation, methylation, phosphorylation, SUMOylation, ubiquitination and ADPribosylation [31]. In many cases, specific histone modifications are associated with transcriptionally permissive or repressive chromatin structure. One well-understood case is acetylation, which is a mark of permissive chromatin. Histone acetylation is regulated by two enzyme families: histone deacetylases (HDACs) and histone acetyltransferases (HATs). An example of the complexity of histone regulation is methylation of lysine 4 or 9 of histone H3 (H3K4 and H3K9), which are marks of permissive and repressive chromatin, respectively. Functionally coherent DNA methylation and histone modifications are often co-imposed by complexes of epigenetic regulators to reinforce repressed or activated states. For example, promoters can be silenced by complexes of DNMT3A/DNMT3B, HDACs and the H3K9 methyltransferase G9A [23]. Another important promoter silencing mechanism independent of DNA methylation is that driven by Polycomb group (PcG) proteins. PcG occupancy As mentioned already, the widely accepted definition of epigenetics as the study of sequence-independent changes in gene expression is based on the assumption that an organism’s genome content remains constant throughout its lifetime. However, enzymes belonging to members of the activation-induced cytidine deaminase (AID/APOBEC) and ADAR (adenosine deaminase) gene families are known to induce specific point mutations in both DNA and RNA (reviewed by Franchini et al. [37]). Indeed, apolipoprotein B editing complex 1 (APOBEC1), which edits the apolipoprotein B RNA, was the first discovered example of an AID/APOBEC deaminase [38]. In DNA, AID/APOBEC enzymes specifically deaminate cytosine into uracil, which if left unrepaired leads to a C:G transition mutation to T:A following DNA replication [37]. Although AID plays a key role in antibody diversification in B cells, increasing evidence implicates AID in active removal of 5-methylcytosine from DNA (see earlier) [37, 39]. Following deamination of 5-methylcytosine (which results in a G:T mismatch) base excision repair enzymes such as thymine DNA glycosylase and methyl-CpG-binding domain protein 4 can remove the thymine and reinsert cytosine. However, the fact that approximately 30 % of all human point mutations are transition mutations in a CpG context, which cannot be explained by spontaneous deamination [40, 41], implies that processes leading to DNA demethylation are likely to contribute to the development of de novo mutations in an individual’s 352, Page 4 of 9 lifetime. To our knowledge, the contribution of these phenomena has not yet been addressed in the context of NR or cellular phenotype switching in atherosclerosis. However, it can be anticipated that next-generation sequencing data will provide interesting insights into the topic. Dedifferentiation of VSMCs to a Synthetic Phenotype Differentiation, proliferation and migration are pivotal cellular functions in the construction of the adult body’s complex structure during embryonic development. Those cellular functions are largely lost in the full-grown adult body. Therefore, an obvious working hypothesis for VSMC dedifferentiation occurring in the atherosclerotic lesion is that at least a partial reactivation of the embryo-specific transcription programme occurs in atherosclerosis-prone vessels. The members of the homeobox (Hox) transcription factor family are important regulators of animal embryonic development and therefore good candidate regulators of VSMC differentiation [42]. Accordingly, class I vertebrate Hox genes have been implicated in atherosclerotic lesion development, yet only a limited number of studies have addressed their relevance in atherosclerosis. Following initial observations that specific Hox members are associated with a VSMC synthetic phenotype, a recent study detected differential expression of a number of Hox genes, particularly Hoxa9, between atherosclerosis-prone and atherosclerosis-resistant aortic portions of 3-month-old, standard-chow-fed apolipoprotein E (apoE)-null mice [43]. The rationale for choosing young apoE-null mice was that they should reveal early or predisposing transcriptional changes, although macrophage-rich atherosclerotic lesions were already visible in the mice used in the study. Importantly, the authors of the study validated the mouse data in rat and pig models. Intriguingly, all differentially expressed Hox gene members were downregulated in the atherosclerosis-prone aortic portion, indicating an atherosclerosis-specific generalized transcriptional repression across the Hox gene cluster, as exemplified by HOXA9 and HOXA10. In the case of HOXA9, the study confirms its role as an important player in vascular biology, particularly the observed antagonism between an endothelium-specific splice variant of HOXA9 (HOXA9EC) and proinflammatory factors such as the transcription factor NF-κB or tumour necrosis factor alpha [44]. The data are consistent with the notion that HOXA9 is a transcriptional activator of the cyclin-dependent kinase inhibitor and tumour suppressor CDKN2A (p16INK4a) [45]. The latter gene maps to 9p21 in humans, a locus robustly linked to cardiovascular risk [46–48]. HOXA10 also displays a predictable expression pattern considering its known role as a transcriptional activator of the proliferation inhibitor CDKN1A (also known as p21; see later) [49]. Still, the data are at odds with Curr Atheroscler Rep (2013) 15:352 previous evidence that two other Hox gene members, HOXB7 and HOXC9, induce VSMC proliferation and are overexpressed in atherosclerotic lesions [42]. In line with the above data, the few genomics-based studies of DNA methylation in vascular tissues suggest that Hox genes are prominent targets for differential methylation in human arteries. At least two studies, one comparing coronary atherosclerotic lesion and lesion-free aortic root tissue, the other comparing aortic and carotid atherosclerotic lesions, yielded several Hox members among the most differentially methylated genes [50, 51]. In particular, epigenetic marks in HOXA11 and HOXA5 were identified as markers of artery-typespecific atherosclerosis, as differential methylation between aortic and carotid lesions was detected in the promoters of those genes [51]. Overall, the involvement of Hox genes in atherosclerotic lesion development is not yet fully understood. Establishing the causal implications of Hox gene epigenetic regulation and differential expression for human atherosclerosis is a challenging issue. First, a comparison between portions of the same artery with differential susceptibility to atherosclerosis in humans is lacking. Second, the results of the aforementioned study by Trigueros-Motos et al. [43] may imply that Hox gene upregulation is an initial event resulting in remodelling of the vasculature to an atherosclerosis-prone tissue, particularly for those Hox members that favour cell proliferation. After this “priming” phase Hox gene upregulation is reversed by proinflammatory stimuli. Alternatively, it is possible that only a small population of VSMCs undergo phenotypic switching and overexpresses Hox genes in early atherosclerosis or prior to lesion formation. Subsequently, those cells are diluted in the lesion mass. Ideally, younger, completely atherosclerosis-free apoE-null mice should be used to verify these hypotheses. A clue to how global DNA methylation profiles may change during VSMC differentiation comes from evidence that during embryo and germ line development, DNA methylation tends to increase during the transition from totipotency to a differentiated state [23]. If ones takes this line of reasoning simply, the dedifferentiation of VSMCs should be associated with a relatively hypomethylated state. This would imply that atherosclerotic lesions are globally hypomethylated in comparison with control tissue. A tendency for hypomethylation in humans and the apoE-null mouse model has been documented by at least two initial studies, but firm evidence on an association between atherosclerosis and loss of DNA methylation is still lacking [52, 53]. To further complicate the issue, global hypermethylation in mice, when artificially imposed by the expression of a bacterial DNMT transgene driven by a smooth muscle cell (SMC)-specific promoter (SMC α-actin), displayed de novo angiogenesis and tumours in SMC-rich organs, suggesting that an increase in DNA methylation may favour VSMC Curr Atheroscler Rep (2013) 15:352 dedifferentiation [54]. The latter hypothesis is indirectly supported by evidence that triglyceride-rich lipoproteins induce global DNA hypermethylation in cultured human macrophages [55]. High-coverage epigenomics analysis of human atherosclerotic lesions is clearly needed to clarify these issues. The participation of histone post-translational modifications in differentiated VSMC marker genes (VSMC-MGs) such as SMC α-actin and SM22-alpha has been studied in detail and was thoroughly reviewed by Alexander and Owens [56•]. This complex relationship can be summarized as follows. The transcriptional co-activator serum response factor binds to specific motifs in VSMC-MG promoters as a complex with myocardin. Work in a cell culture model of VSMC differentiation established that transcriptional activation by serum response factor–myocardin depends on local chromatin remodelling to a permissive, acetylated histonerich state by retinoic acid [57]. These effects on chromatin structure could be facilitated by a direct interaction between myocardin and HATs. In vivo data show that the involvement of histone acetylation in the maintenance of VSMC differentiation is complex and likely gene-specific, since the HDAC inhibitors Scriptaid and trichostatin A inhibit proliferation and neointima formation in animal models by inhibiting the positive regulator of cell proliferation cyclin D1 (reviewed by Findeisen et al. [58•]). As for drivers of VSMC-MG repression and VSMC dedifferentiation, the transcription factor Krüppel-like factor 4 (KLF4) is a much studied candidate as it is upregulated in atherosclerosis and can repress myocardin and VSMC-MG expression (reviewed in [56•]). Furthermore, KLF4 is a plausible main trigger of VSMC dedifferentiation, since it is one of a small group of prominent pluripotency inducers [59]. Yet, two unexpected findings indicate that there is still much to be learnt about KLF4 activity. First, conditional KLF4 inactivation in mice induces VSMC proliferation [60]. Second, the aforementioned suppressive effects of HDAC inhibitors on neointima formation were associated with KLF4 induction [61]. Interestingly, KLF4 was shown to induce the cyclin D1 inhibitor CDKN1A [62]. Several explanations can be put forward to explain these inconsistencies. As often pointed out, findings in cell culture models and in vivo are often different if not opposed [56•]. Furthermore, the phenotype of KLF4 conditional knockout described by Yoshida et al. [60] in mice is complex, as neointima formation is preceded by an initial delay of SMC dedifferentiation. This phenotype may be explained by attributing a temporally restricted “priming” function to KLF4 in VSMC phenotype switching which subsequently gives way to a different regulatory mechanism, akin to the hypothesized scenario for Hox genes (see earlier). A caveat is that KLF4 is downregulated during monocyte-tomacrophage differentiation; therefore, macrophage differentiation and atherogenesis may be accelerated in KLF4- Page 5 of 9, 352 conditional mutant mice [24•]. Another important issue is that manipulation of an individual transcription factor or chromatin regulator expression is likely to exert global effects on several genes and thus produce complex downstream responses. Evidence for the importance of lncRNAs in atherosclerosis has been provided by the analysis of the human 9p21 locus. This locus show a robust genetic association with atherosclerosis that is independent of traditional risk factors (see earlier). This region harbours the lncRNA antisense ncRNA in the INK4 locus (ANRIL), also known as CDKN2B antisense RNA (CDKN2BAS). Several ANRIL transcripts, including circular transcripts, have been identified and are associated with atherosclerosis risk [63–65] (reviewed by Holdt and Tuepser [66]). Interestingly, small interfering RNA mediated targeted silencing of two different exons differentially affects the expression of atherosclerosisrelated genes in VSMCs, suggesting independent functions of ANRIL splice forms [67]. Furthermore, ANRIL is involved in epigenetic silencing of the cyclin-dependent kinase inhibitor genes CDKN2B (encoding p15INK4b) and CDKN2A (see earlier), both of which are well-known regulators of cell proliferation and senescence, via its recruitment of PcG proteins [68, 69]. Additional ncRNAs that mediate cellular responses to angiotensin in VSMCs, including an lncRNA that is responsible for the production of two miRNAs (miR221 and miR-222) implicated in cell proliferation, have recently been described [70]. Likewise, several miRNAs have been shown to participate in vascular remodelling events (reviewed by Nazari-Jahantigh et al. [71]). A summary of selected mechanisms that regulate VSMC differentiation is presented in Fig. 1. VSMCs Switch to a Macrophage-Like Phenotype and to an Osteoblast-Like Phenotype The participation of epigenetic mechanisms in the transdifferentiation of VSMCs to macrophage-like or osteoblast-like cells is a relatively scarcely studied topic. The peroxisome-proliferator-activated receptor γ coactivator 1α, an activator of HAT p300, confers a proinflammatory phenotype to VSMCs in response to high glucose concentrations [72]. As for transdifferentiation of VSMCs to osteoblast-like cells, calcification is a common feature of human atherosclerosis and is associated with hyperphosphataemia. Cell-culture-based and aortic-tissueculture-based models of high-phosphate-concentration-induced conversion of VSMCs to osteoblast-like cells revealed that promoter methylation and repression of the differentiated VSMC-specific SM22-alpha gene accompany the induction of osteoblast markers, although the detailed mechanisms are not yet understood [73]. An unexplored domain of 352, Page 6 of 9 Curr Atheroscler Rep (2013) 15:352 Fig. 1 Overview of epigenetic mechanisms involved in vascular smooth muscle cell (VSMC) phenotype switching. In differentiated VSMCs (lower part, left to right) myocardin (MYOCD) in a complex with serum response factor (SRF) homodimers keeps differentiated VSMC marker genes (VSMC-MG) actively transcribed. Histone acetyltransferase (HAT) activity induced by retinoic acid (RA) seeds acetyl groups (Ac) in VSMC-MG promoters and facilitates transcriptional co-activation by MYOCD [56•]. Cell proliferation is restricted by the activation of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2A by Krüppel-like factor 4 (KLF4) and the transcription factors HOXA9 and HOXA10 [45, 49, 62]. In dedifferentiated VSMCs (upper part, left to right) the long non-coding RNA ANRIL favours cell proliferation by silencing the tumour suppressor CDKN2B through recruitment of Polycomb group (PcG) proteins [68, 69]. Repressive methylation marks are seeded at the promoter of the VSMC-MC SM22alpha gene (black lollypops) [73]. Furthermore, hypermethylation is possibly a genome-wide mark of the atherosclerotic lesion [54]. KLF4 plays a complex role in these events, as it can promote cell proliferation by silencing MYOCD and various VSMC-MGs. This effect is accomplished by releasing HATs from VSMC-MG promoters [56•]. HOXB7 acts as a driver of VSMC proliferation [42] by activating targets, including possibly the epidermal growth factor receptor gene (EGFR), which is upregulated in dedifferentiated VSMCs [79]. Arrows and crosses in gene boxes indicate active and repressed transcriptional states, respectively epigenetic regulation is the generation of osteoclasts in atherosclerosis, which may be part of a regressive process with potential therapeutic implications. It has recently been shown that lesion osteoclasts originate from the recruitment and NR of circulating monocyte/macrophages, thus uncovering yet another layer of vascular cell plasticity in which lesion macrophage functions are not limited to sustaining inflammation [74]. the same hurdles that are limiting transcription-factor-based therapy. With the exception of peroxisome-proliferatoractivated receptors and oestrogen receptors, transcriptionfactor-based drugs have been difficult to design, as shown by the fact that they account for only approximately 10 % of prescribed medications [75]. One further problem is identifying critical epigenetic marks to be targeted. Quantitatively robust epigenetic changes associated with pathological phenotypes in a population of cells or in a lesion are obvious candidates. On the other hand, less abundant changes that are specific for a critical subpopulation of cells—for example selected VSMCs that secrete locally active proinflammatory factors—may be disregarded for falling below a predefined significance threshold value. This problem is particularly relevant in the light of the cellular heterogeneity of the atherosclerotic lesion and may not be completely eliminated even by purifying homogeneous cell populations. Indeed, Conclusions Any detection of epigenetic changes in a gene promoter or any other genomic sequence is an expected finding. Epigenetic regulation is as ubiquitous as the regulation of promoter activity by transcription factors. This means that the design of effective epigenetic therapies is likely to face Curr Atheroscler Rep (2013) 15:352 very recent single-cell RNA-seq data have revealed that cellto-cell variation in gene expression is remarkably high even in supposedly homogeneous populations, implying that epigenetic changes are likely to show a comparable level of variability [76]. In addition, only an extraordinary technological advance will allow the targeting of the desired epigenetic marks to or the erasing of unfavourable ones from specific sequences in specific cells. One preliminary, successful example is the effectiveness of a methylated oligonucleotide in silencing the insulin-like growth factor II gene in a murine model of hepatocarcinoma [77]. Another promising area that is very relevant to the topic of this review is the attempt to target specific HDAC classes to modify the histone signature of only a set of critical genes [61, 78]. Clearly, exciting research lies ahead to find creative solutions to understand the pathobiological and therapeutic implications of vascular cell NR. Conflict of Interest Silvio Zaina, Maria del Pilar Valencia-Morales, Fabiola E. Tristán-Flores and Gertrud Lund declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. References Papers of particular interest, published recently, have been highlighted as: • Of importance 1. Gurdon JB, Melton DA. Nuclear reprogramming in cells. Science. 2008;322:1811–5. 2. De Robertis EM, Gurdon JB. Gene activation in somatic nuclei after injection into amphibian oocytes. Proc Natl Acad Sci USA. 1977;74:2470–4. 3. • Ma T, Xie M, Laurent T, Ding S. Progress in the reprogramming of somatic cells. Circ Res. 2013;112:562–74. 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