Role of microRNAs in endothelial function.

MicroRNAs (miRNAs) are a class of short, single-stranded, noncoding RNAs that modulate a variety of biological functions as negative regulators of gene expression. Post-transcriptional regulation by miRNAs is crucial in many aspects including cell proliferation and death. They play important regulatory roles in endothelial cell function, which are significant for the treatment of endothelial dysfunction and related metabolic disorders. This review summarizes current insights into the functions of miRNAs, focusing specifically on the regulation of endothelial function. MiRNAs are endogenous, small, noncoding, single-stranded RNAs of ˜22 nucleotides that regulate gene expression by targeting mRNAs for cleavage or translational repression.1 There is growing evidence that they modulate a variety of biological functions in cell proliferation, differentiation, apoptosis, and angiogenesis.1–3 In endothelial cells (ECs), several miRNAs have been identified in the control of new blood vessel proliferation, including miR-126, miR-296, and the miR-17-92 cluster.4–6 In addition, miR-217 has been shown to modulate ECs senescence.7 These findings suggest that miRNAs play important roles in the regulation of endothelial function and angiogenesis. Ecs, which form the inner lining of blood vessels, regulate vascular homeostasis. The proliferation, sprouting, and migration of ECs play critical roles in the formation of new blood vessels (angiogenesis).8 Therefore, miRNAs induction in ECs has potential therapeutic applications for perfusion following ischemia. On the other hand, inhibition of angiogenesis is a novel therapeutic strategy for combating tumor growth. Furthermore, endothelial dysfunction precedes the development of atherosclerosis and contributes to the development of lesions in cardiovascular diseases. Here, we reviewed the roles of specific miRNAs in the regulation of endothelial function. BIOLOGY OF MIRNAS MiRNAs are a class of highly conserved, single-stranded, noncoding small RNAs.1 These endogenously expressed molecules are transcribed in the nucleus as long primary transcripts (pri-miRNAs). These pri-miRNAs, which contain the active miRNAs in their characteristic stem-loop structures, are further processed by RNase III endonuclease Drosha into shorter precursor species (pre-miRNAs) of approximately 70 nucleotides (nts). Pre-miRNAs are transported to the cytoplasm by exportin-5 and subsequently processed by cytosolic enzyme dicer into the 20- to 24-nt mature miRNAs, which form complexes with the RNA-induced silencing complex (RISC).9–13 After maturation, they enter the RNA interference pathway and regulate gene expression at the post-transcriptional level by translational repression or mRNA degradation (Figure 1).9Figure 1.: MiRNA biogenesis and function. The primary transcripts of miRNAs, called pri-miRNAs, are transcribed from miRNAs genes. The RNase Drosha processes the pri-miRNAs into shorter precursor species, called pre-miRNAs, which are exported from the nucleus by exportin 5. In the cytoplasm, pre-miRNA is processed by Dicer into an miRNA duplex. Incorporated into the RNA-induced silencing complex (RISC), the mature miRNA negatively regulates gene expression at the post-transcriptional level by translational repression or mRNA degradation.REGULATION OF MIRNAS EXPRESSION IN ECS PRO-ANGIOGENIC MICRORNAS MiR-126 MiR-126 is an endothelium-specific miRNA that is highly expressed in human ECs.14 In mammals, miR-126 is located in intron 7 of epidermal growth factor (EGF)-like domain 7, an ECs-derived secreted protein essential for proper vascular development.15 It has many physiological functions, including regulating angiogenic signaling and vascular integrity. In vivo, miR-126 deletion in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, owing to loss of vascular integrity and defects in ECs proliferation, migration, and angiogenesis. MiR-126 knockdown in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development.4,16 In addition, miR-126 has been shown to regulate vascular cell adhesion molecule 1 (VCAM-1), providing direct evidence for the contribution of miR-126 to proper ECs function.17–19 Further insights into mechanisms of regulation were gained through in-depth analysis of miR-126 target genes. MiR-126 regulates ECs angiogenic activity in response to angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, targeting Sprouty-related, EVH1 domain-containing protein-1 (SPRED1),20 a negative regulator of extracellualr signal-regulated protein kinase signaling, and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2),21,22 an upstream effector of protein kinase B.22 Increased expression of SPRED-1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown.4,16,23 MiR-126 also regulates vascular integrity in zebrafish by targeting p21-activated kinase1 (pak-1), an important cytoskeletal regulator involved in regulating cell-cell junction turnover.24 In addition, it inhibits atherosclerosis in mice by mediating the atheroprotective effects of endothelial apoptotic bodies. It exerts the atheroprotective effects by targeting the regulator of G-protein signaling 16 (RGS16). RGS16 can induce C-X-C chemokine receptor 4 expression in ECs, which in turn increases the production of stromal cell-derived factor-1 (SDF-1), whose anti-inflammatory and plaque-stabilizing properties play a role in atherosclerosis.25,26 A latest study also indicated that miR-126 can regulate vasculogenesis by modulating the endothelial expression of SDF-1, which plays a major role in the migration and recruitment, and contributes to neovascularization.27,28 MiR-130a MiR-130a expression was detected in proliferating ECs. MiR-130a contains important regulatory sequences for the growth arrest-specific homeobox (GAX) 3′-untranslated region (UTR), which control the down-regulation of GAX and homeobox (HOX) A5 expression in response to mitogens, pro-angiogenic factors, and proinflammatory factors.29 GAX, which is expressed in both vascular smooth muscle cells and ECs, has many properties suggestive of a major regulator of ECs phenotype in response to pro-angiogenic and anti-angiogenic signals.30 It is expressed at its highest level in quiescent ECs. Down-regulation of GAX is prominent when ECs are exposed to serum or pro-angiogenic factors, causing inhibition of cell growth and activation. By down-regulating the expression of GAX and HOX-A5, miR-130a antagonizes the anti-angiogenic activity of GAX and HOX-A5 in ECs.29 MiR-210, miR-16 and miR-424 MiR-210 is a key player in cell responses to hypoxia, modulating ECs survival, migration, and differentiation.31 Under hypoxic conditions, the molecular mechanisms of response to oxygen deprivation are extremely complex, which is a key role being played by hypoxia-inducible factor (HIF). Several studies have shown that HIF is critical for the hypoxic induction of miR-210.31–33 In normoxic ECs, miR-210 over-expression stimulated VEGF-driven cell migration and the formation of capillary-like structures on Matrigel, whereas its blockade decreased cell migration caused by VEGF and inhibited the formation of capillary-like structures.31 Similarly, another study demonstrated that miR-210 could be involved in targeting the VEGF signaling pathway to regulate angiogenesis after renal ischemia/reperfusion injury.34 These effects were mediated, at least in part, by the direct inhibition of the receptor-tyrosine kinase ligand Ephrin A3 (EFN-A3).31,34 EFN-A3 is a direct target of miR-210 in hypoxia and its modulation by miR-210 has significant functional consequences. The expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis.31 In hypoxic ECs culture, regulation of miR-210 expression affects cell survival, migration, and differentiation. Furthermore, other miR-210 targets have been recently identified including E2F transcription factor (E2F) 3, DNA Repair And Recombination Protein, activin A receptor, type 1B, MAX dimerization protein, caspase-8-associated protein 2, fibroblast growth factor receptor-like 1, and HOX-A1 and -A9,35–41 suggesting multiple roles for this miRNA in the cellular adaptation to hypoxia. In addition, VEGF, VEGF receptor 2 (VEGFR-2), and fibroblast growth factor receptor 1 (FGFR-1) were identified and validated as targets of miR-16 and miR-424 in ECs, which suggests that miR-16 and miR-424 play important roles in regulating cell-intrinsic angiogenic activity of ECs.42 MiR-378 MiR-378 expression plays a key role in enhancing angiogenesis, tumor cell survival, and tumor growth, enhancing cell survival, reducing caspase-3 activity, and promoting tumor growth and angiogenesis by targeting two suppressors, Sufu (suppressor of fused) and Fus-1 (tumor suppressor candidate 2, TUSC2).43 Expression of luciferase constructs containing the target sites in Sufu and Fus-1 was repressed by miR-378. Cotransfection experiments showed that miR-378 suppressed Sufu and Fus-1 expression. Transfection of Sufu and Fus-1 constructs reversed the function of miR-378, suggesting an important role of miR-378 in cell survival. These findings indicate that Sufu and Fus-1-mediated pathways are essential for miR-378-enhanced angiogenesis and cell survival.43 MiR-296 MiR-296 plays a major role in angiogenesis.6 Angiogenic growth factors promote its expression in cultured primary human brain microvascular ECs. Down- and up-regulation of miR-296 resulted in the inhibition and induction, respectively, of morphological characteristics associated with angiogenesis in human ECs. Sequence-specific inhibition of miR-296 resulted in decreased neo-vascularization of tumors in mice, and up-regulation of miR-296 expression was demonstrated in tumor blood vessels. Altogether, these results support a role of increased miR-296 levels in promoting angiogenesis in tumors.6 Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR-2 and platelet-derived growth factor receptor-β.6 Moreover, EGF could induce miR-296, suggesting a complex growth factor-growth factor receptor cross-talk mechanism that elevates the levels of miR-296.6 Let-7 family and miR-27 Let-7 family and miR-27b have been identified as factors modulating angiogenesis in ECs.44 Inhibition of let-7f and miR-27b in ECs reduced sprout formation, indicating that they promote angiogenesis by targeting anti-angiogenic genes. Several members of the let-7 family are highly expressed in human umbilical vein cells (HUVECs).44,45 Let-7b participates in endothelial angiogenesis by regulating the expression of the anti-angiogenic factor tissue inhibitor of metalloproteinase 1 (TIMP-1).46 In silico prediction of potential target genes revealed that the let-7 miRNA family may regulate the expression of thrombospondin 1 (TSP-1), which is an endogenous angiogenesis inhibitor; however, inhibition of let-7f induced only a minor, non-significant, increase in TSP-1 expression.44 As described by Zhou et al,47 miR-27 enhanced angiogenesis by promoting angiogenic signaling through targeting Sprouty-2 and Sema 6. MiR-27b was found to correlate with pulsatile shear-induced ECs growth arrest by targeting E2F-1, and KH-type splicing regulatory protein was also proved to be a new target,48 but other targets of miR-27b remain to be determined.49 MiR-17-92 cluster The miR-17-92 cluster components were among the first miRNAs linked to tumor angiogenesis, and they could involve in the response to irradiation.50 In the human genome, this cluster encodes miRNAs, such as miR-17, miR-18a, miR-19a/b, miR-20a, and miR-92a, which are highly expressed in several tumors.51 The miR-17-92 cluster is significantly up-regulated in myc-induced tumors and over-expression of the entire cluster increases tumor angiogenesis via a paracrine mechanism.52 This pro-angiogenic function has been attributed to down-regulation of the anti-angiogenic molecules, TSP-1 and connective tissue growth factor, which are predicted targets for repression by the cluster. In particular, miR-18 preferentially suppresses CTGF expression, whereas miR-19 targets the potent angiogenesis-inhibitor TSP-1.52 In ECs cultures, over-expression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a enhanced ECs sprout formation. Inhibition of miR-17 and miR-20a in vivo significantly increased the number of perfused vessels in Matrigel plugs, whereas inhibitors that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor vessel growth.53 Further mechanistic studies indicated that miR-17/20 target several pro-angiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In addition, miR17-5p down-regulated TIMP-1 expression and activity, participating in ECs proliferation and motility.46 In summary, miR-17/20 exhibit cell-intrinsic anti-angiogenic activity in ECs. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis, indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.53 MiR-92a regulates the growth of new blood vessels. Its over-expression in ECs blocked angiogenesis. Conversely, its inhibition enhanced blood vessel growth and improved the functional recovery of damaged tissue.54 It has been reported that the integrin subunit alpha-5, a crucial regulator of ECs functions, is a directly regulated target of miR-92a in ECs.54 As reported by Tréguer et al,55 the expression of some miR-17-92 cluster members, miR-17, -18, and -19, was increased during the induction of endothelial differentiation of pluripotent stem cells, while the expression of other members, miR-92a and the primary miR-17-92 transcript, was decreased. Their work also indicated that it is not the members but the miR-17-92 cluster that has a significant impact on the endothelial differentiation. In conclusion, the miR-17-92 cluster is differentially involved in the regulation of angiogenesis. More evidence is needed, however, to evaluate the probability of targeting specific members of this family for improving therapeutic angiogenesis. ECS SENESCENCE-RELATED MIRNAS MiR-217 and miR-34a It has been proposed that ECs senescence is involved in endothelial dysfunction and atherogenesis.56 A recent study indicated that over-expression of miR-217 promotes endothelial senescence and its inhibition in old ECs ultimately reduces senescence and increases angiogenic activity.7 Similarly, miR-34a over-expression leads to significantly increased ECs senescence and impedes angiogenesis.57 Intriguingly, it has been demonstrated that both miRNAs target silent information regulator 1 (SirT-1).7,57,58 SirT-1 plays a critical role in regulating cell cycle, senescence, apoptosis, and metabolism, has been shown to exert protective effects against endothelial dysfunction, and plays a key role in angiogenesis.59,60 Recently, a synthetic lipid-lowering agent named atorvastatin was found to be associated with up-regulation of SirT-1 expression via inhibition of miR-34a.61 The study elucidated a new mechanism by which statin therapy could improve endothelial dysfunction. In a word, miR-217 and miR-34a are endogenous inhibitors of SirT-1, which promotes endothelial senescence. MiR-23a and miR-24 Moreover, miR-23a and miR-24 exert the same effects as miR-217 and miR-34a do. However, the differences are that miR-23a may be involved in ECs apoptosis through regulation of the caspase-7 and serine/threonine kinase 4-caspase-3 pathways and miR-24 induces endothelial cell apoptosis by targeting the endothelium-enriched globin transcription factor binding protein 2 (GATA2) and the p21-activated kinase 4 (PAK-4).62,63 This makes them potential targets for therapeutic manipulation to prevent endothelial dysfunction in metabolic disorders. ECS PROLIFERATION-RELATED MIRNAS Recently, miR-100 has been shown to be strongly expressed in ECs and to modulate proliferation, tube formation, and sprouting activity in these cells.64 Over-expression of miR-100 resulted in inhibition of both ECs network formation and cell sprouting. In contrast, inhibition of miR-100 resulted in increased endothelial network formation and a longer total sprout length by targeting the mammalian target of rapamycin (mTOR).64 MTOR, a master regulator of cell growth and proliferation, controls specific aspects of cellular metabolism by regulating metabolic gene expression.65 MiR-100 is a down-regulated miRNA in ECs. It has inhibitory effects on neovascularization in vitro and in vivo through regulation of its target gene mTOR. ANTI-ANGIOGENIC AND INFLAMMATION-RELATED MICRORNAS MiR-221/miR-222 MiR-221 and miR-222 belong to the same family and have been identified in HUVECs, participating in regulation of angiogenesis and homeostasis.50 They regulate expression of c-kit, the receptor for stem cell factor (SCF), a key inhibitor of ECs migration, proliferation, and angiogenesis.45 MiR-221 and miR-222 modulate the angiogenic activity of SCF and the level of its receptor c-kit; miR-221/222-transfected cells can no longer form tubes or heal wounds in response to SCF.45 Exposure to high glucose levels induced expression of miR-221 but reduced expression of c-kit, causing ECs dysfunction and impairing ECs migration. Incubation with the anti-miR-221 oligonucleotide (AMO-221) reduced expression of miR-221 and restored c-kit protein expression in HUVECs. Furthermore, AMO-221 treatment abolished the inhibitory effect of high glucose exposure on HUVECs transmigration. These findings suggest that manipulation of the miR-221-c-kit pathway may offer a novel strategy for treating vascular dysfunction.66 In addition, miR-222/221 indirectly regulates endothelial nitric oxide synthase (eNOS) expression via gene expression, translational efficiency or post-translational mechanisms in human ECs.67 An earlier study showed that up-regulation of miR-221/222 in coronary artery disease patients might contribute to the observed dysfunction and reduced regenerative capacity of endothelial progenitor cells (EPCs), which can normally differentiate into endothelial lineage cells.68 In addition to the c-kit target, P27KIP1 (encode cyclin-dependent kinase inhibitor 1B) and P57KIP2 (encode cyclin-dependent kinase inhibitor 1C) are two novel targets of miR-221/222, through which miR-221/222 performs the cell-specific functions in ECs. 69,70 Interestingly, a recent study71 investigated the novel contribution of miR-221/222 to inflammation-mediated neoangiogenesis and their potential involvement in atherosclerotic intraplaque neovascularization. MiR-126, -221, -222, and -296 were down-modulated in ECs exposed to inflammatory stimuli, but only miR-222 was involved in inflammation-mediated vascular remodeling. In addition, signal transducer and activator of transcription 5A (STAT-5A) was identified as a novel target of miR-222, with a negative correlation between miR-222 and STAT-5A expression in ECs from advanced neovascularized atherosclerotic lesions.71 Furthermore, miR-221/222 has been shown to target E26 transformation-specific sequence factor 1 (Ets-1), which indirectly regulates the expression of several ECs inflammatory molecules and therefore reduces HUVECs migration, attenuates the adhesion of Jurkat T cells to activated HUVECs, and extends the role of miR-221/222 in endothelial inflammation.72 In summary, these findings indicated a critical role of miR-221/222 in endothelial function, presenting possible therapeutic targets in vascular diseases. MiR-155 It is widely accepted that inflammation contributes to multiple vascular diseases. MiR-155, an inflammation-related miRNA, was detected in ECs by in situ hybridization.73 Current studies presented evidence that miR-155 participates in endothelial inflammation and migration by targeting angiotensin II type 1 receptor (AT1R) or Ets-1 in Ang II-stimulated HUVECs.72–75 Functional AT1Rs are presented in ECs. They have multiple roles, which include regulating the concentration of intracellular free calcium, mediating the release of vasodilator molecules, and promoting eNOS expression and vasodilation.76–78 In addition, Ets-1, an important endothelial transcription factor, has been shown to regulate endothelial inflammation, angiogenesis, and vascular remodeling.79 Its downstream targets, including vascular cell adhesion molecule 1, monocyte chemotactic protein-1 and Fms-related tyrosine kinase 1, were up-regulated markedly in AngII-activated HUVECs.72 By targeting both AT1R and Ets-1, miR-155 regulated endothelial inflammation.72,73 Moreover, miR-155 decreases the number of Jurkat T cells adhering to AngII-activated HUVECs and AngII-induced HUVECs migration.72 Collectively, miR-155 participates in endothelial inflammation and migration by targeting AT1R or the transcription factor Ets-1 in ECs. ENOS has been found to be another direct target of miR-155 as reported by Sun et al.80 The 3'-UTR of eNOS mRNA is the very binding sites to decrease eNOS expression and NO production in HUVECs. Inflammatory cytokines, such as tumor necrosis factor-α, are believed to account for the up-regulation of miR-155, and miR-155 is a potential versatile therapeutic target for inflammatory disease.80, 81 MiR-21 and miR-200 MiR-21 acts as a negative regulator of angiogenesis by down-regulating its targets, both RhoB82 and peroxisome proliferators-activated receptor-alpha (PPAR-α) in HUVECs,83 and High mobility group A2 (Hmg-A2) in EPCs. Besides miR-21, miR-200b acts also as a negative regulator of angiogenesis by down-regulating its targets, Ets-1 in human microvascular ECs,84 VEGF in HUVECs,85,86 and VEGFR-2 and GATA2 in human dermal microvascular ECs.87 MiR-200c, another miR-200 family member, is up-regulated in HUVECs induced by H2O2 and promotes cell apoptosis by targeting ZEB1.88 DISCUSSION Increasing evidence indicates the importance of miRNAs in regulating ECs functions and the formation of new blood vessels (Table 1), together with a key role in endothelial dysfunction and vascular disease. Because the function of each miRNA is thought to regulate hundreds of mRNAs and each miRNA is mediated by the modulation of specific mRNA targets,89 the identification of new miRNA targets represents an important step in elucidating miRNA function. Identification of miRNAs and their targets and a better understanding of the mechanisms that regulate endothelial miRNA expression and processing are essential for miRNA-based therapy.Table 1: . Endothelial function-regulatory miRNAs and their targetsIn conclusion, the findings of the current studies provide novel insights into the complex regulation of endothelial function, and further studies are needed to analyze the complex interactions between endothelial-specific miRNAs and their targets during disease.