Noncoding RNAs in diabetes vascular complications.

YJMCC-07975; No. of pages: 9; 4C: 5 Journal of Molecular and Cellular Cardiology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

Review article

Noncoding RNAs in diabetes vascular complications Cristina Beltrami a, Timothy G. Angelini b, Costanza Emanueli a,c,⁎ a b c

National Heart and Lung Institute, Imperial College of London, London, UK Royal Surrey County Hospital -NHS Trust, Guilford, UK Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, UK

a r t i c l e

i n f o

Article history: Received 28 October 2014 Received in revised form 18 November 2014 Accepted 5 December 2014 Available online xxxx Keywords: MicroRNAs Long noncoding RNAs Diabetes Vascular cells Bone marrow-derived cells Extracellular vesicles Biomarkers

a b s t r a c t Diabetes mellitus is the most common metabolic disorder and is recognised as a dominant health threat of our time. Diabetes induces a widespread damage of the macro- and microvasculature in different organs and tissues and disrupts the endogenous vascular repair mechanisms, thus causing diffuse and severe complications. Moreover, diabetic patients respond poorly to surgical interventions aiming to “revascularise” (i.e., to restore blood flow supply) the ischemic myocardium or lower limbs. The molecular causes underpinning diabetes vascular complications are still underappreciated and druggable molecular targets for therapeutic interventions have not yet clearly emerged. Moreover, diabetes itself and diabetes complications are often silent killers, requiring new prognostic, diagnostic and predictive biomarkers for use in the clinical practice. Noncoding RNA (ncRNAs) are emerging as new fundamental regulators of gene expression. The small microRNAs (miRNAs, miRs) have opened the field capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers. More recently, long ncRNAs (lncRNAs) have started to be actively investigated, leading to first exciting reports, which further suggest their important and yet largely unexplored contribution to vascular physiology and disease. This review introduces the different ncRNA types and focuses at the ncRNA roles in diabetes vascular complications. Furthermore, we discuss the potential value of ncRNAs as clinical biomarkers, and we examine the possibilities for therapeutic intervention targeting ncRNs in diabetes. This article is part of a Special Issue titled: Non-coding RNAs. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . MicroRNAs . . . . . . . . . . . . . . . . . . . 2.1. MiRNA expressed in vascular cells . . . . . . 2.2. miRNA expressed in bone marrow cells . . . 2.3. Extracellular miRNAs and extracellular vesicles 2.4. MiRNAs as biomarkers in the diabetes setting Long noncoding RNAs . . . . . . . . . . . . . . 3.1. LincRNAs . . . . . . . . . . . . . . . . . 3.2. Natural antisense (NATs) lncRNAs . . . . . . 3.3. Other lncRNA forms . . . . . . . . . . . .

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Abbreviations:(AGE), advancedglycation end product; (Ang II),angiotensinII; (AS),antisense; (Ago), argonaute;(BM), bonemarrow; (CAC),circulatingangiogeniccells; (CAD), coronary artery disease; (CLI), critical limb ischemia; (DM), diabetes mellitus; (EC), endothelial cell; (EPC), endothelial progenitor cells; (EV), extracellular vesicles; (FGF), fibroblast growth factor; (GAWS), genome-wide association study; (GDM), gestational diabetes; (HG), high glucose; (HUVEC), human umbilical vein endothelial cell; (IGF-1), insulin-like growth factor-1; (IIGF-R), IGF receptor; (LincRNA), intergenic RNAs; (IL-6), interleukin 6; (KLF 15), Kruppel-like transcription factor 15; (LncRNA), long ncRNA; (MALAT-1), metastasis-associated lung adenocarcinoma transcript-1; (MP), microparticles; (MiRNAs MiRs), microRNA; (MCP-I), monocyte chemotactic protein I; (MNC), mononuclear cell; (NATs), natural antisense; (NC), noncoding; (NT), nucleotide; (nc RNA), noncoding RNA; (POL II), polymerase II; (pre-MiRNAs), precursor MiRNA; (pri-MiRNAs), primary MiRNAs; (PAC), proangiogenic circulatory cells; (siRNA), silencing RNA; (SNP), single nucleotide polymorphisms; (TF), transcription factors; (TGF), tumour growth factor; (T1DM), type 1 diabetes mellitus; (T2DM), type 2 diabetes mellitus; (VEGF-1), vascular endothelial growth factor A; (VSMC), vascular smooth muscle cells; (3′-UTR), 3′-untranslated region; (5′-UTR), 5′-untranslated region. ⁎ Corresponding author at: Bristol Heart Institute, Bristol Royal Infirmary-Level 7, University of Bristol, Bristol BS8 2HW, UK. Tel.: +44 117 342 3512. E-mail address: [email protected] (C. Emanueli).

http://dx.doi.org/10.1016/j.yjmcc.2014.12.014 0022-2828/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Beltrami C, et al, Noncoding RNAs in diabetes vascular complications, J Mol Cell Cardiol (2014), http://dx.doi.org/ 10.1016/j.yjmcc.2014.12.014

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C. Beltrami et al. / Journal of Molecular and Cellular Cardiology xxx (2014) xxx–xxx

3.4. LncRNA expressed in vascular cells . . . . . . . 3.5. Other lncRNA linked to diabetes . . . . . . . . . 4. Open questions, translational perspectives and conclusions Funding . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder, characterised by hyperglycemia, which can appear at different phases of life, including in utero, and results in multisystem complications. The most common forms of DM are type 1 and type 2 DM (T1DM and T2DM). T1DM, which accounts for 5–10% of all diabetic cases, is caused by the destruction of the insulin-producing pancreatic beta-cells as a result of a complex process where genetic and environmental factors lead to an autoimmune response. In contrast, the prevalent T2DM presents as peripheral insulin resistance, mostly caused by reduced insulin sensitivity in skeletal muscle, adipose tissue and liver, resulting in a compensatory hypersecretion of insulin that precedes beta-cell exhaustion and the reduction in beta-cell secretory function. DM may also occur as a consequence of pregnancy (gestational DM [GDM]), genetic defects of the beta-cells, genetic defects in insulin action, endocrinopathies and use of drugs and infections [1]. It is estimated that 347 million people worldwide are affected by DM and epidemiological studies indicate that the global prevalence of DM will double by 2030. Recent evidence has shown that high blood glucose is the third leading risk factor for global mortality, with 5.8% of deaths globally [2]. More than 50 % of people with DM die of cardiovascular disease, making it a major cause of morbidity and mortality in diabetic patients. Macrovascular and microvascular complications are the most significant and complex consequences of DM and spread to different organs and tissues, especially the heart, kidney, lower limbs, brain and eyes, putting the diabetic patients at an increased risk of coronary artery disease (CAD), renal failure, critical limb ischemia (CLI), stroke and blindness [1, 3–7]. DM not only causes vascular dysfunction it also impairs the endogenous repair mechanisms able to counteract vascular damage, including angiogenesis and vascular progenitor cell responses to tissue ischemia and arterial endothelial denudation [8, 9]. As a consequence, the clinical outcome of diabetic patients after surgical or balloon-mediated revascularisation is considerably worse [10–12]. In addition to this, diabetics with CLI have a much higher risk of minor and major limb amputation [11, 12]. Molecular causes of DM vascular disease have been a key area of investigation for several years, with the aim of developing better therapeutic targets and discovering clinical biomarkers enabling early diagnosis in those at high risk of diabetes and track the development and/or progression of diabetic complications. To date, cardiovascular diabetic complications remain unmet clinical needs, calling for novel and revolutionary research. This review focuses on noncoding (nc) RNAs as possible new therapeutic targets to combat the insurgence and evolution of diabetic vascular complications and as possible biomarkers enabling to identify and monitor such complications in diabetic patients. The recent advancement in DNA and RNA-sequencing techniques have led to the understanding that around 98% of the human genome contains regions of DNA which code for RNA molecules unable to produce proteins and hence termed ncRNAs [13]. Far from being “junk RNA,” the ncRNAs are proving to be very powerful regulators of gene expression, acting at both transcriptional and post-transcriptional level. Noncoding RNAs include many different subtypes, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and enhancer-like RNAs (eRNAs) [14]. The ncRNAs are arbitrarily defined accordingly to their size, as small ncRNAs and long ncRNAs (lncRNAs, with sequences longer than 200 nucleotides [nt]). MicroRNAs (miRNAs, miRs) are the

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most popular small ncRNAs. Here, we will review miRNA and lncRNAs for their regulatory roles in vascular function, with special attention to their contribution to diabetic complications. Finally, we will discuss the potential of ncRNAs as new therapeutic targets and clinical biomarkers to be employed in diabetic patients. 2. MicroRNAs The pathways of miRNAs biogenesis and function have been widely reviewed by ourselves and others [15–29] and will be only briefly recapitulated here. The production of miRNAs is a multistep process. miRNAs are typically transcribed by RNA polymerase II (Pol II) as primary miRNAs (pri-miRNAs), which are cleaved to precursor miRNAs (premiRNAs) and finally to mature forms, containing a single RNA filament of a 20-22 nt. MiRNAs mediate post-transcriptional gene silencing by guiding Argonaute (Ago) proteins to messenger RNA (mRNA) targets [30]. The canonical mRNA targeting pathway by a miRNA involves the recognition and binding between the miRNA “seed sequence” (8 nt) in the 5′-untranslated region (5′-UTR) and “miRNA binding site(s)” located in the 3′-untranslated region (3′-UTR) of the mRNA. This miRNAmRNA interaction happens with either perfect or near perfect match via a Watson-Crick base-paring mechanism. Owing to their ability of each miRNA to silence hundreds of different target genes, it has been estimated that miRNAs regulate gene expression of more than 60% of the mRNAs. Moreover one mRNA can be targeted by more than one miRNA, thus adding complexity to the regulatory networks. In addition, new mechanisms of miRNA regulation have been described [31–34], and new literature suggests that the consequences of miRNA–target interactions might go beyond repression of gene expression (reviewed in [30]). 2.1. MiRNA expressed in vascular cells MiRNAs are produced by every human cell. The initial concept of tissue- and cell-specific expression of miRNAs has been refuted with the acquisition of new knowledge. Notwithstanding, it is still true that some miRNAs are enriched in certain cells and tissues. For example, using RNA deep sequencing, Voellenkle et al. have shown that miR-21 and miR-126 totalled almost 40% of all miRNAs expressed by human umbilical vein endothelial cells (HUVECs), the most common endothelial cell (EC) model employed for in vitro research [35]. However, miR21 is also widely expressed in cardiac fibroblasts, cardiomyocytes, kidney cells and cancer cells [36–38]. Moreover, miR-126 expression expands to platelets [39] and several cancer cells [39]. Other miRNAs which are widely expressed in the cardiovascular system are miR-1, -24, -133a/b, -208a/b and -499 and miR-145. However, with the possible exception of miR-208a, which might be purely cardiac, the other miRNAs are also expressed in other tissues under healthy and cancerous conditions [40]. This provides further issues with the use of miRNAs as a diagnostic tool and therapeutic targets. As miRNAs are found in most cells throughout the body, this means they are unlikely to be sensitive, specific and easy to target without eliciting side effects at distant tissues. DM has been proposed to alter the expression and function of many of the aforementioned miRNAs. miR-126 was one of the first miRNAs to be found to have altered circulating concentrations in T2DM [41]. miR-21, which is highly involved with fibrosis, has been shown to



mediate fibrotic responses in the context of diabetic nephropathy [42]. MiR-133 is also affected by DM and it mediates some of its responses. Furthermore, miR-133a is down-regulated in the heart of T1DM mice [43], in association with increased expression of its direct target, tumour growth factor-ß (TGF-ß), leading to the development of cardiac fibrosis [44]. On the other hand, overexpression of miR-133 has a direct role in cardiac glucose transport by targeting both glucose transporter type 4 (GLUT4) and Kruppel-like transcription factor 15 (KLF15), an inducer of GLUT4 expression [43]. The miR-143/145 gene cluster has a critical role during VSMC differentiation and in the reversion of the VSMC differentiation phenotype that occurs during vascular disease [45]. Gene knockout of the miR-143/-145 cluster provokes structural modifications of the mouse aorta because of an incomplete differentiation of VSMCs [45]. Recently, it was shown that miR-143/145 are increased in the saphenous vein-SMCs from T2DM patients [46] and that this was accompanied with increased cell area and reduced proliferation, which could be “corrected” by inhibition of the miRNAs [46]. We speculate that this miR-143/-145-induced anti-proliferative effect on VSMCs is not pathogenic in the context of vein graft (the surgical procedure which requires the use of saphenous vein for bypass grafting of occluded coronaries or limb arteries). In fact, increased VSMC proliferation is cause of vein graft stenosis and failure. In line, the transduction of miR-145 into a rabbit vein used for bypass grafting prevented vein graft disease by regulation of VSMC phenotype [47]. On the other side, miR-143/ 145 deficiency was reported to attenuate the progression of plaque formation in a mouse model of atherosclerosis [48], indicating that more research is necessary to clarify the role on this miRNA cluster in vascular disease. MiRNAs have been shown to contribute to the vascular complications of DM. Wang et al. demonstrated different miRNAs expression in ECs treated with high glucose (HG) concentration [49]. The authors showed that miR-320 was highly expressed in diabetic myocardial microvascular ECs, which was associated with impaired functional capabilities of the ECs. By contrast, miR-320 inhibition promoted angiogenesis [49]. The negative effects of miR-320 are presumably mediated by its direct targeting of vascular endothelial growth factor A (VEGF-A), fibroblast growth factors (FGFs), insulin-like growth factor 1 (IGF-1) and the IGF receptor IGF-1R [50]. Our group demonstrated that miR-503 has a pivotal role in DM-induced endothelial dysfunction. MiR-503 expression is increased in ECs cultured in HG and reduced supplements (to mimic DM and ischemia-associated starvation). Lentivirusmediated overexpression of miR-503 in HUVECs reduced cell proliferation, migration, networking capacities and the expression of the cell cycle regulators cdc25A and cyclin E1. Accordingly, miR-503 inhibition re-established normal functional capacities of ECs cultured under HG/ low growth factor conditions [51]. Moreover, increased miR-503 was also detected in total ischemic muscles and in ECs extracted from the ischemic muscles of T1DM mice as well as in muscle samples of T2DM patients undergoing major amputation for CLI [51]. Most important, local miR-503 inhibition resulted in improved post-ischemic blood flow recovery and angiogenesis in a mouse model of T1DM and unilateral limb ischemia [51], thus strongly suggesting miR-503 as a therapeutic target in ischemia associated with DM. Another miRNA involved in angiogenesis and targeted by DM in ECs is miR-221. Exposure of HUVECs to HG increases miR-221 expression [52]. Recently, increased level of miR-221 and miR-222 were detected in the internal mammary artery (aka, internal thoracic aorta) of subjects with T2DM [53]. However, different results were found by Togliatto et al. [54]. The reduction of miR-221 expression was associated with impaired angiogenesis and inhibition of cell cycle progression due to HG and high levels of advanced glycation end product in HUVECs [54]. Interestingly, in the rat genome, miR-221 neighbours is co-transcribed with an lncRNA (Lnc-Ang362, vide infra in the lncRNA section) [55]. In cultured vascular cells, HG treatment was also found to up-regulate miR-125b, with a consequent down-regulation of one of the miR-125b targets, the histone-lysine N-methyltransferase Suv39h1. In microvascular SMCs from a mouse

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T2DM model, Suv39h1 was shown to bind to the promoter of proinflammatory genes, such as interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1), to enhance their expression [56, 57]. In separate studies, miR-125b has been proved a key regulator of apoptosis by binding p53 [58, 59]. From all the above, it is evident that miRNAs are linked with diabetic vascular alterations. However, this subject needs further investigation. Table 1 summarises the miRNAs implicated in diabetes vascular complications. 2.2. miRNA expressed in bone marrow cells Cardiovascular homeostasis and repair is supported by different populations of stem and progenitor cells, which either reside locally in cardiovascular tissues or in stem cell-enriched compartments, principally the bone marrow (BM) [60]. BM-derived mononuclear cell (MNC) populations that are capable of “regenerative” capacities circulate in the peripheral blood. As a group, these different cell populations were initially baptised as “endothelial progenitor cells” (“EPCs”). However, some “EPC” types have later been renamed as proangiogenic circulating cells (PACs), circulating angiogenic cells (CACs) or similar compromising names after controversies emerged in regard to their capacity to differentiate to ECs [60]. It is, however, widely accepted that the “old EPCs” retain the capacity of promoting vascular growth and repair at least by paracrine actions, namely, by secreting a series of “therapeutic factors.” By these means, these circulating cell populations represent an endogenous repair mechanism which, to a certain extent, provides a buffering mechanism for altered microenvironments, namely, tissue ischemia (by inducing angiogenesis) or intravascular injury (by promoting the rendothelisation of the arterial intimal layer). The BM “vascular regenerative” cells (which for simplicity we identify as “EPCs”) are contained in specialised niches and released by the BM in a regulated manner. However, DM impairs the number and quality of these BM-derived circulating cells [9]. Both laboratory bases and clinical studies have demonstrated that hyperglycemia induces alterations to “EPCs” [9]. The reduction and dysfunction of these proangiogenic cells correlates with the occurrence and severity of microvascular and macrovascular complications, suggesting a close mechanistic link between “EPC” dysfunction and impaired vascular function/repair in DM (reviewed in [61]). Moreover, working on a mouse T1DM model, we acquired the first evidence that DM-induced microangiopathy in the BM disrupts the integrity of the BM, adding to the loss of the regulation in “EPC” release [61]. We have recently confirmed these concepts working on human clinical samples from patients undergoing either amputation for CLI or hip replacement surgery. This allowed us to provide new clinically relevant, anatomical and molecular evidence for the damaging effects of DM on the human BM, comprising microvascular rarefaction and shortage of CD34-positive progenitor cells, possibly attributable to the activation of apoptosis [62]. In fact, in DM, increased apoptosis of BM CD34-positive cells was associated with up-regulation and nuclear localisation of the proapoptotic factor FOXO3a and induction of the FOXO3a targets p21 and p27 (kip1). Of high note in the setting of this review, miR-155, which promotes cell survival through inhibition of FOXO3a, was down-regulated in diabetic patient-derived BM CD34positive cells and inversely correlated with FOXO3a levels. The effect of T2DM on the anatomical and molecular end points was confirmed when considering background covariates. Furthermore, the transcriptional changes induced by T2DM were mimicked by in vitro exposure of healthy BM CD34-positive cells to HG and, it could be prevented by transfecting the cells with a pre-miR-155 [62]. The clinical benefit achieved by autologous transplantation of BM cells and BM-derived circulating cells have been investigated in a series of early clinical trials, mainly with a focus on post-ischemic vascular repair. The outcome of these trials shows promise but suggests the necessity of the refinement at multiple levels, including the use of more powerful cells. It may be that future treatments lies in allogenic


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Table 1 MiRNAs that are expressed by the vasculature or proangiogenic cells and modulated by diabetes mellitus. miRNAs

Expressed in (among vascular cells)

Up/down-regulation

Targets

Function regulated

References

miR-15a/16

PACs

Up

Apoptosis, angiogenesis, migration

[52]

miR-21

ECs

Up

Apoptosis, angiogenesis, proliferation, migration

[25–27]

miR-24

ECs

Up

Apoptosis, angiogenesis, proliferation, migration

[30,106,107]

miR-125b

ECs VSMC

Up

NF-kB signalling and inflammation Increase macrophage activation Regulation expression IL-6 and MCP-1 Apoptosis

[47 48]

miR-126

ECs Platelets BM-derived CD34+cells

Down

Angiogenesis

[28,66 68]

miR-143/-145

VSMC

Up

SMC differentiation and proliferation

[34–37]

miR-155

BM CD34+cells

Down

Apoptosis

[51]

miR-221/-222

ECs SMC ECs ECs

Up

VEGF-A AKT3 Spry1 PDCD4 PTEN RhoB Sorbin, SORBS, PDLIM52 GATA2 PAK4 RASA1 H2AFX eNOS TNFAIP3 IRF4 Suv39h1 p53 SPRED1 VCAM-1 VEGF IGF-2 Dlk1 KLF α-SMA myosin VI FOXO3a p21 p27 ckit, p27kip1, p57kip2

Angiogenesis, Cell cycle

[41–43]

Up Up

VEGF, FGF, IGF-1, IGF-1-R cdc25, CCNE1

Angiogenesis, proliferation migration Angiogenesis, proliferation migration

[38,39] [40]

miR-320 miR-503

α-SMA, alpha smooth muscle actin; AKT3, v-akt murine thymoma viral oncogene homolog 3; BM, bone marrow; CCNE1, cyclin E1; cdc25, cell division cycle 25; Dlk1, Protein delta homolog 1; ECs, endothelial cells; eNOS, endothelial nitric oxide synthase 3, FGF, fibroblast growth factor; FOXO3a, Forkhead box O3; GATA2, GATA binding protein 2; H2AFX, H2A histone family, member X; IGF-1, insulin growth factor 1; IGF-2, insulin growth factor 2; IGF-1-R, insulin growth factor 1 receptor; IRF4, Interferon regulatory factor 4; PACs, proangiogenic cells; KLF, Kruppel-like factor 4; PAK4, serine/threoninep21-activated kinase; PDCD4, programmed cell death protein 4; PDLIM52, PDZ and LIM domain 5; PTEN, phosphatase and tensin homolog; p27kip1, cyclin-dependent kinase inhibitor 1B; p57Kip2, cyclin-dependent kinase inhibitor 1C; p21,cyclin-dependent kinase inhibitor p21; p53, tumour protein p53; RASA1, RAS p21 protein activator (GTPase activating protein) 1; RhoB, Ras homolog gene family, member B; SMC, smooth muscle cells; SORB, Sorbin and SH3 domain containing 1; SPRED1, sprouty-related, EVH1 domain containing 1; Spry1, protein sprouty homolog 1; Suv39h1, histone-lysine N-methyltransferase SUV39H1; TNFAIP3, tumor necrosis factor, alpha-induced protein 3; VEGF, vascular endothelial growth factor; VEGF-A, vascular endothelial growth factor A; VCAM-1, vascular cell adhesion protein 1; VSMC, vascular smooth muscle cells.

transplantation with of-the-shell pluripotent stem cell-derived products. However, there are potential safety and immune reaction risks that come with this approach and they must be resolved before moving to clinical trials. Consequently, research into autologous cell therapy is still on the agenda. It is conceivable that miRNAs affect the regenerative potential of BM-derived cells and that miRNA targeting can be used therapeutically. Hence, we began investigating the expression of selected miRNA in PACs from CLI patients with/out T2DM and noticed that some miRNAs were differently expressed in patients-derived PACs [63]. We focussed on miR-15a and miR-16, which are clustered together and share the seed sequence with the aforementioned miR-503 [64]. We found that miR-15a/16 elicited proapoptotic and anti-angiogenic function. Conversely, the use of specific miRNA inhibitor in PACs could improve their functional performance in vitro and in vivo. In fact, the ex vivo engineered PACs were noted to have a higher regenerative potential following their transplantation in an immunocompromised limb ischemia mouse model [63]. This allows the speculation that ex vivo miRNA targeting could be used to empower a variety of cells type candidates for autologous transplantation. 2.3. Extracellular miRNAs and extracellular vesicles Functionally competent miRNAs can be actively released by their parent cells and be absorbed by neighbouring cells. Consequently, extracellular miRNAs are currently investigated as gene expression regulators in cell-to-cell communication. Moreover, miRNA circulate in the blood in a protected status, conferring them with resilience to degradation. This led to the hypothesis that extracellular miRNAs might serve as new noninvasive biomarkers. Indeed, extracellular miRNAs are present in virtually any kind of biological fluid and are resistant to RNAse

activity and to hostile environmental conditions [65, 66]. This miRNA resilience derives from the miRNA being released by the cells in two main ways. Either, as embedded extracellular vesicles (EVs), such as microparticles (MPs), exosomes [67] and apoptotic bodies [68] or conjugated with RNA binding proteins [69] or lipoprotein complexes [70]. EVs are able to transfer miRNAs, protein, and mRNA between cells [71, 72] and take part in several biological functions, such as angiogenesis, tumour cell invasion, cell proliferation or immune response to neighbouring or distant cells [73]. The regulation of these processes is still subject of investigation and may involve the direct activation of the receptor on the surface of different cells by protein binding or lipid ligands, followed by the release of the effector into recipient cells as well as fusion of the EV membrane with the plasmalemma of the recipient cell [67, 74, 75]. Many cell types, including cardiovascular cells, have already been shown able to secrete exosomes [26, 76, 77] and MPs [77]. Diabetes may affect both the quantity and quality of EVs and miRNAs. Indeed, circulating EC-derived MPs are increased in T2DM patients [78]. Additionally, MPs released from ECs cultured under standard conditions promote vascular repair via transfer of miR-126; by contrast, MPs from ECs exposed in vitro to HG to mimic hyperglycemia show reduced miR-126 content and impaired regenerative potential [77]. In line with this, the Landmesser group showed that circulating proangiogenic cells are enriched with miR-126, and by transfer of this miRNA, they can elicit regenerative properties. However, when cells of the same type are prepared from the blood of T2DM subjects, the molecular and functional profiles are impaired [79]. Moreover, it was shown that exosomes released by rat cardiac myocytes present differences in their miRNA cargo and in their functional properties depending on the health status of the donor rats. Cardiomyocytes isolated from normal rats produced



exosomes able to promote angiogenesis-favouring responses (cell proliferation, migration and tube formation) by ECs. Conversely, exosomes released by cardiac myocytes of T2DM rats contained higher miR-320 levels and by transfer of this miRNA elicited the opposite response in ECs [80]. Taken together, the above evidences suggest that extracellular miRNA and EVs might play an important, but yet largely unexplored, role in diabetic cardiovascular complications. 2.4. MiRNAs as biomarkers in the diabetes setting Because of their resilience and the fact that many miRNAs are enriched in particular tissues (and hence their presence in the blood could flag the health status of their tissue source), miRNAs are regarded as potential new clinical biomarkers. Indeed, among the cardiovascular medical and surgical communities, there is great expectation for miRNAs as novel biomarkers with diagnostic, prognostic and predictive value. However, we are still at a primordial phase of these investigations and so far no miRNA has been validated to be superior to the traditional biomarkers in use. In the setting of DM, there is a real need to improve the prediction, detection and monitoring of vascular complications in patients with DM or with pre-DM. We have earlier referred to the pioneer work by Zampetaki et al. [41], who identified a plasma miRNA signature for DM that includes loss of endothelial miR-126. These data, which have been later confirmed by an independent laboratory [81], necessitates further validation in different and larger cohorts of diabetic patients and controls allowing investigation into possible differences in baseline miR-126 levels due to, for example, varying genetic and environmental backgrounds. In fact a measurable reduction of miR-126 could represent a noninvasive biomarker for both pre-diabetes and also diabetes-induced endothelial dysfunction. We have also identified increased miR-503 and miR-15a and -16 in the circulation of CLI patients with T2DM. For miR-503, patients were recruited at the moment they underwent a major amputation [51], while the miR-15/16 study was performed in patients with CLI, when the limb was still salvageable [63]. Moreover, because T2DM patients are at particular risk of developing acute and late complications following angioplasty, or bypass grafting, it is of paramount importance to investigate new markers allowing for a better risk stratification peri-interventionally. Importantly, we have provided the first evidence that extracellular miRNAs could

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be used as predictive biomarkers in T2DM patients undergoing revascularisation for CLI [63]. We studied a population of 122 T2DM subjects, with a follow-up at 12 months after revascularisation and found that baseline serum miR-15a and miR-16 levels positively correlated with amputation and restenosis at follow-up [63]. More genome-wide-based discovery research using clinical samples from well characterised patients and validation of the putative miRNA “biomarker” on larger patients' cohorts and biobanks is imperative for evolving to the next stage, which will hopefully see miRNA biomarkers changing the clinical practice. 3. Long noncoding RNAs The human genome contains information for many thousands of lncRNAs [14], which have not been studied to the same level of miRNAs, yet. LncRNAs are thought to contribute in various biological processes such as transcription, translation, splicing and intracellular and extracellular trafficking [82]. LncRNAs present a structural and functional heterogeneity. LncRNA classification has been based on their position in respect to the protein-coding gene (see Fig. 1). On this basis, lncRNAs can be intergenic (lincRNAs), antisense (AS), intronic or divergent [83]. LncRNAs can function as important modulators of the expression of protein-coding genes located nearby (in cis, i.e., “on this side” regulation) or at distance (trans regulation). Moreover, lncRNAs may be used for various tasks, including post-transcriptional regulation, organisation of protein complexes, cell-to-cell signalling and allosteric regulation of proteins. The lncRNAs can participate in the activation of the transcription of different genes by cooperating in opening the chromatin structures to make it accessible to the transcriptional machinery. This event may involve both a recruitment of transcription factors (TFs) or even hybridisation to them or to their binding site to enhance the process [84, 85]. However, lncRNAs can also repress gene transcription by binding to a specific locus or to other elements of the transcription machinery by overlapping with TF binding sites. Furthermore, lncRNAs can be cleaved into shorter silencing RNAs (siRNAs) and therefore degrade the related mRNA transcripts [84–88]. A good review of lncRNA biology can be found in [89]. Below, we briefly introduce lncRNA classes before moving to describe the vascular role of lncRNAs and their potential link with diabetes and its vascular complications.

Fig. 1. LncRNAs classification. Schematic diagram representing the LncRNA (violet box) transcript associated with the corresponding protein-coding gene (green box). Intronic lncRNA: the lncRNA sequence is derived completely from within an intron of another protein-coding gene. Intergenic (lincRNA) lncRNA: the lncRNA sequence do not overlap with any protein-coding gene and they have an independent transcriptional unit. Natural antisense transcript (NAT) lncRNA: the lncRNA sequence initiate inside or at both the 3′ and 5′ UTR ends of a proteincoding gene with an opposite direction. NAT may also overlap with the corresponding sense mRNA. Divergent lncRNA: the lncRNA sequence is located on the opposite strand from a protein-coding gene whose transcript is initiated b1000 base pairs away.


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Table 2 summarises the lncRNAs described expressed in vascular cells and their known functions. 3.1. LincRNAs Long lincRNAs are lncRNAs that are strictly intergenic and do not overlap with known protein-coding genes. LincRNAs have an independent transcriptional unit and are primarily transcribed by RNA polymerase II. The RNA product can be 2,000–20,000 nt long [90], and it can present polyadenylation, poly(A) tails at 3′ end. The promoter region of these loci is highly conserved and the sequences have been preserved throughout evolution [91, 92]. The expression patterns of lincRNA suggest that they might have a role in different biological processes, such as cell cycle regulation, innate immunity, stem cell pluripotency and also establishment of chromatin state by modifying proteins involved in repressing the gene expression at specific loci [92]. Examples of lincRNAs are metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which is also expressed in ECs (vide infra) [93], and lincRNA-p21, which is present in VSMCs (vide infra) [94]. 3.2. Natural antisense (NATs) lncRNAs The natural antisense (NATs) lncRNAs initiate inside or at both the 3′ and 5′ UTR ends of a protein-coding gene. The NAT transcription happens in the opposite direction to the protein-coding gene. A NAT may also overlap with the corresponding sense mRNA, and its expression is not related to the corresponding sense mRNA, suggesting that the NTA and the mRNA have independent transcription and regulation. NATs can be located in both the cytoplasm and nucleus and play multiple cellular roles, such as transcriptional regulation, mRNA stability, splicing and epigenetic control [90, 95]. An example of NAT is ANRIL (also known as CDKN2BAS), which is encoded by the NK4b-ARF-INK4a locus at Chromosome 9p21 (in human). Genome-wide association studies (GAWS) have identified this region as a hot spot, associated with cardiovascular disease and T2DM [96, 97]. In fact, single nucleotide polymorphisms (SNP) mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including CAD and T2DM [97]. In cultured VSMCs, ANRIL has been shown to modulate the expression of gene sets directly involved in atherosclerosis [98]. 3.3. Other lncRNA forms The intronic lncRNAs are located and encoded inside an intron of a protein-coding gene and they do not overlap with any exon. Their expression profiling appears to be regulated together with their corresponding protein-coding genes [99]. They exhibit their functions in different intracellular locations [100]. The divergent lncRNA sequence faces in the opposite direction to the protein-coding gene and has a cut-off distance of less than 1,000 base pairs [83]. It was also shown

that lncRNAs can originate from transcriptionally active pseudogenes [101] and even from the mitochondrial genome [102]. Using an lncRNA array-based screening of human plasma samples, Kumarswamy et al. [103] found that the bona fide mitochondrial long noncoding RNA uc022bqs.1 (LIPCAR) and other lncRNAs of mitochondrial origins had their expression up-regulated in patients who developed negative left ventricle remodeling after an acute myocardial infarct. However, it is difficult to discriminate the nuclear or mitochondrial origin of mitochondrial RNAs, due to the duplication of the mitochondrial DNA in the genome [104]. LIPCAR expression was positively associate with a higher risk of cardiovascular death for patients with heart failure [103]. 3.4. LncRNA expressed in vascular cells Reports of lncRNA expressed in the vasculature are finally emerging. HUVECs express the aforementioned MALAT1 prevalently in their nuclear fraction and the still unexplored linc00657 and linc00493 in their cytoplasm. Hypoxia increases MALAT1 and appears to mediate proangiogenic response in vitro and in mouse angiogenesis models [93]. It is not known if MALAT1 is modulated by DM. However, this would be an interesting area of investigation since DM reportedly compromises reparative angiogenesis. LncRNAs have also been found to be expressed in VSMCs, where their functional roles and possible links to diseases are emerging. The Miano group performed an RNA-sequencing-based genome-wide analysis of lncRNAs expressed in cultured human coronary artery SMCs and identified 31 new lncRNAs, including SENCR (for smooth muscle and endothelial cell–enriched migration/differentiation-associated lncRNA), which they have then further characterised [105]. SENCR is transcribed in the antisense orientation from within the first intron of FLI1, but there is no overlap between SENCR and the FLI1 exonic sequences. Consequently, SENCR is an antisense lncRNA, but not a NAT. SENCR is cytoplasmic and seems to stabilise the VSMC contractile phenotype and to inhibit VSMC migration [105]. SENCR has not yet been studied in the context of DM. However, as hyperglycemia-induced growth of VSMCs is a characteristic features of the cardiovascular complications of DM, SENCR might possibly be implicated in this pathogenic pathway [106]. Similarly, the recently identified lincRNA-p21 might have an unexplored link with diabetic vascular complications. In fact, lincRNA-p21 reportedly controls p53 signalling to repress proliferation and induce apoptosis of both VSMCs and macrophages [94]. LincRNA-p21 was found to be down-regulated in atherosclerotic plaques of ApoE gene knockout mice and lincRNA-p21 inhibition promoted neointimal hyperplasia in a rodent carotid artery injury model [94]. Increased angiotensin II (Ang II) is highly involved in the deleterious action of DM on the vasculature, including the aforementioned excessive proliferation of VSMCs [105]. Consequently, it is extremely interesting that Ang II has been shown to affect the lncRNA expressional profile in VSMCs [55]. Using transcriptome and epigenome profiling, Leung et al. have identified several

Table 2 LncRNAs that are expressed by the vasculature. lncRNA

Class

Models

Function

Reference

MALAT1

LincRNA

ECs

[82]

LipcRNAp21

LincRNA

VSMC Macrophages

ANRIL

NAT

VSMC Blood MI patients

Mediate proangiogenic response ECs proliferation Enhance p53 activity and apoptosis Target the promoter/enhancers of genes such as Puma, Bax, Noxa and MDM2 Represses expression of oncosuppressor INK4B, ARF, INK4a

HAS2-AS1 SENCR

NAT Antisense

LIPCAR LncAng362

VSMC ECs Plasma VSMC

Hyluronan synthesis regulation (induction of HAS2 transcription) Inhibit of VSMC migration Stabilise VSMC contractile phenotype Possible biomarker of cardiac remodelling and HF Reduce VSMC proliferation

[83] [86,87] [95] [93,94] [92] [44]

ARF, ADP-ribosylation factor; Bax, BCL2-associated X protein; EC, endothelial cell; HAS2, hyaluronan synthase 2; HF, heart failure;INK4a cyclin-dependent kinase inhibitor 2A; INK4B, cyclin-dependent kinase 4 inhibitor B; MDM2, mouse double minute 2 homolog; MI, myocardial infarction; NAT, natural antisense; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; PRC2, polycomb repressive complexes 2; Puma, p53 up-regulated modulator of apoptosis; VSMC, vascular smooth muscle cells.



new lncRNAs and lncRNA which are differently expressed in response to Ang II in rat VSMCs. The majority of the genomic loci of these novel transcripts were enriched with histone marks (H3K4me3 and H3K26me3), which are usually found at actively transcribed regions. Knockdown of the aforementioned Lnc-Ang362 reduced VSMC proliferation. Hence, we can suggest that some of the newly identified noncoding transcripts could be exploited as novel therapeutic targets for Ang II associated cardiovascular diseases [55]. A possible limitation of this study is that it was performed on nonhuman cells. It is known that, as opposed to miRNAs, lncRNAs are not often conserved between species. Consequently, we would suggest that expensive and long experiments, such as those referred to here, should preferentially be developed on human cells and, when available, human clinical samples. A recent study performed on human aortic SMCs and atherectomy samples of human carotid arteries at different stages of the disease suggests another possible link between DM and lncRNA regulation in the vasculature [107]. Hyaluronan (HA) accumulation into artery walls is involved in cardiovascular diseases and in diabetic vascular complications. Vigetti et al. have found that HA synthesis is regulated via a NAT for HA Synthase 2 (HAS2-AS1). In particular, HAS2-AS1 induced transcription of HAS2 via protein O-GlcNAcylation. This is also relevant because of O-GlcNAcylation contributes to vascular calcification in DM [108]. 3.5. Other lncRNA linked to diabetes Pancreatic islet cells produce insulin and glucagon, two hormonal regulators of glucose homeostasis. Interestingly, the lncRNA H19 is involved in GDM-associated alteration of islet structure and function as well as in the intergenerational transmission of GDM [109]. Global lncRNA screening approaches conducted by Moran et al. systematically interrogated the lncRNA transcriptome in human pancreatic beta-cells and reported expressional changes in islet-specific lncRNAs during progenitor commitment, following stimulation with glucose and in response to T2DM [110]. The data strongly suggest a pathophysiological role of lncRNAs in the pancreatic tissues, which might be linked with DM [110, 111]. Insulin resistance constitutes a key step in T2DM development. Consequently, it is an exciting observation that insulin and IGF-1 signaling influences lncRNA expression (e.g., of the lncRNA CRNDE) [111, 112]. We expect that in the near future work by several groups including ours will provide a clearer picture on the role of vascular lncRNAs and how they influence cardiovascular risk factors, diseases and DMinduced complications. 4. Open questions, translational perspectives and conclusions The diabetic vascular complications represent unmet clinical needs. Obviously, the attention should primarily focus on achieving glycemic control by insulin supplementation and/or other anti-diabetic drugs, diet adjustments and exercise. Notwithstanding, the Epidemiological study of Diabetes Intervention and Complications (EDIC) and the Diabetes Control Complications Trial (DCCT) provided evidence that even with tight glycemic control, vascular complications continue to operate for over five years after restoration of normoglycemia, possibly due to the persistence of epigenetic or otherwise long-lasting molecular marks (reviewed in [113]). It is therefore crucial to better understand the molecular changes underlining DM and its vascular complications and to subsequently unravel the potential of new molecular therapies for preventing or reducing diabetic complications. In just a few years of intense research, miRNAs have been shown to have a significant influence on vascular function and stem and progenitor cell behavior. However, the studies performed specifically in the field of diabetic vascular complications are still too scarce to propose a translational pathway. We should remember that so far miRNA therapeutics have not yet reached first-in-man cardiovascular trials and that too often in the past sensationalism with poorly reasoned initial

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clinical attempts have resulted in long-lasting damage to the field of research. We propose that a series of well-designed prospective observational clinical studies with the collection of biopsies (and when available surgical leftover samples) for RNA sequencing should be developed to identify the master regulator miRNAs and lncRNAs in the molecular networks leading to diabetic complications. This reverse translational approach integrated by bioinformatics and system biology should then be validated in different cohorts of diabetic patients, as well as in cells and animal models (including large animals). Research initiated in the lab and validated in mouse models has too often disappointed at the level of clinical trials resulting in the pharmaceutical and medical communities becoming dubious of developing new therapeutics from basic scientific ideas. However, the power of genome-wide analyses, such as RNA sequencing, developed directly on patientderived samples and GWAS, could give new impetus to molecular medicine. The importance of work on human cells and samples is particularly true for lncRNAs, which, contrarily from the miRNAs, are not considered well conserved among species. We have described several approaches available to modulate the miRNA expression in a recent review of ours [114]. Importantly from clinical translation, clinical grade miRNA inhibitors have been already developed by companies and, encouragingly, one noncardiovascular clinical trial (ClinicalTrials.gov number, NCT01200420 Funded by Santaris Pharma) based on the inhibition of miR-122 (using “miravirsen”) in patients with chronic Hepatitis C virus genotype 1 infection showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance and safety at phase 2a) [115]. Today, too little is known on lncRNA biology and their vascular function to propose them as new therapeutic targets. Notwithstanding, we predict an exponential interest in these new ncRNAs by the biomedical community and that in a few years their basic biology and contribution to diseases could be better understood. For the moment we note that systemic antisense oligonucleotide (ASO)-mediated inhibition of disease-associated lncRNAs was able to produce therapeutic effects in mouse models of degenerative muscular diseases [116]. Further questions are posed by the possibility that different classes of lncRNAs are released extracellularly and hence measurable in biological fluids as markers of disease. However, the literature on this topic is scarce and also contradictory, which may be a result of the different classes/lncRNAs and their intracellular location. However, preliminary evidence suggests that lncRNAs can be found in exosomes released by cancer cells [117]. Moreover, patients who had experienced a myocardial infarction showed detectable LIPCAR expressional changes in plasma, which, has also been associated with negative left ventricle remodelling at follow-up [103]. It is may be too soon to say if cell- and tissue-specific lncRNAs exist, but if they do, they could represent better circulating markers of vascular disease in comparison with miRNAs. Funding The work was funded by the Leducq transatlantic network in vascular microRNAs (MIRVAD) and the National Health Research Institute (NIHR) Bristol Cardiovascular Biomedical Research Unit (BRU) (both to CE). Disclosures The authors report no conflicts of interests. The views expressed are those of the Authors and not necessarily those of the NHS, the NIHR or the Department of Health. References [1] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2005;28(Suppl. 1):S37–42. [2] World Health Organization. Global health risks: mortality and burden of disease attributable to selected major risks; 2009.


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