A Primer on the Role of MicroRNAs in Endothelial Biology and Vascular Disease

A Primer on the Role of MicroRNAs in Endothelial Biology and Vascular Disease Jason E. Fish, PhD Summary: Endothelial cells are highly proliferative and motile during vascular development. However, as blood vessels mature and stabilize the endothelial lining becomes quiescent, and cell– cell interactions among endothelial cells generate a stable barrier between the blood and tissue. Rather than simply functioning as an inert barrier, endothelial cells constantly sense and respond to environmental cues. Activation of the endothelium can promote the loss of cell– cell adhesion and an increase in the motility and proliferation of the endothelium. This process is requisite for tissue repair, but also plays a role in vascular pathogenesis and is especially relevant to kidney injury. The molecular mechanisms that facilitate these phenotypic alterations are only partially understood. Recent work has shown that microRNAs can modulate endothelial phenotype. These new insights have shed light on the complex mechanisms that endothelial cells use to respond to environmental stimuli. This review addresses the known roles that microRNAs play in controlling angiogenic and inflammatory signals in endothelial cells, and illustrates that microRNAs are important modulators of endothelial function in vascular disease, and therefore represent promising therapeutic targets. Semin Nephrol 32:167-175 © 2012 Elsevier Inc. All rights reserved. Keywords: Endothelium, microRNA, vascular disease, inflammation, gene regulation

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ndothelial cells play key roles in the pathogenesis of multiple human diseases including atherosclerosis, coronary artery disease, renal artery stenosis, stroke, sepsis, macular degeneration, diabetes, and cancer, among others. This review focuses on the known and proposed roles for microRNAs in regulating vascular function, with particular emphasis on microRNAs expressed in the endothelium. Much work also has shed light on the crucial roles that microRNAs play in smooth muscle biology, and their contribution to vascular disease,1-8 but this will not be discussed in depth here. Although this review does not focus solely on the renal vasculature, the topics covered here are relevant to our understanding of the contribution of microRNAs to vascular formation and function in a variety of organs, including the kidney. This article concentrates on the roles of microRNAs in three aspects of vascular biology: angiogenesis, vascular repair, and inflammation. This article also provides suggestions for how microRNA expression may be used as a biomarker of vascular disease, and how microRNAs might be targeted therDivision of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; The Heart and Stroke/ Richard Lewar Centre of Excellence, Toronto, Ontario, Canada. Financial disclosure and conflict of interest statements: Work in the laboratory of JEF is funded by the Heart and Stroke Foundation of Ontario, grant NA-7282. Address reprint requests to Jason Fish, PhD, Toronto General Research Institute, University Health Network, MaRS Centre, Toronto Medical Discovery Tower, 101 College St, Suite 3-308, Toronto, Ontario M5G 1L7, Canada. E-mail: [email protected] 0270-9295/ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2012.02.003

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apeutically to treat various diseases that involve the vasculature. More than 700 microRNAs are predicted to be encoded in the human genome, however, only a small number of these microRNAs have been characterized functionally. Considering that many microRNAs are expressed in a dynamic and/or cell-restricted fashion, future efforts to clone novel microRNAs from samples isolated from specific subsets of cells at different developmental stages, or in normal or pathologic adult tissues, may unearth an even greater repertoire of microRNAs. Recent evidence also suggests that other classes of noncoding RNAs, including long intergenic noncoding RNAs, play functional roles in cellular biology,9 but their mechanisms of action are poorly understood. It has been estimated that nearly a third of all messenger RNAs (mRNAs) are targeted by microRNAs,10 and experimental validation has shown that a single microRNA can directly modulate the expression of hundreds of genes,11 suggesting that microRNAs have a widespread effect on the proteome. Clearly, many microRNAs can target the mRNA for a single protein coding gene. In general, microRNAs bind to the 3’ untranslated regions (UTRs) of target mRNAs and negatively regulate the stability and/or translation of the mRNA.11 Although most studies have implicated microRNAs in the repression of gene expression, microRNAs also can enhance translation,5,12,13 but the mechanisms involved, and how often this occurs, are not well understood. In general, the seed region (bases 2-7, numbered from the 5’-end) of a microRNA binds with complete complementarity to a target mRNA, with weaker microRNA/mRNA interactions often occurring near the 3’-end of the microRNA. In silico approaches for identifying microRNA targets therefore have focused 167

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on searching for seed matches present in the 3’ UTRs of mRNAs. This undoubtedly results in many false-positive targets, and these approaches also may miss genuine microRNA targets that do not conform to the classic seed-match model of microRNA/mRNA interaction.14 Additional criteria, such as the evolutionary conservation of target sites across multiple species,15 as well as the accessibility of potential target sites in the 3’ UTR,16,17 also are useful in defining real targets. However, nonconserved microRNA targets have been validated, and it is difficult to accurately predict accessibility in vivo. The identification of bonafide microRNA targets is therefore quite challenging. MicroRNA expression is regulated dynamically in the cell, and this regulation occurs at multiple levels. MicroRNA transcripts can be generated by RNA polymerase II–mediated transcription of intergenic regions, or microRNAs can be processed from the introns or exons of host mRNAs. Transcription of microRNAs can be dynamic,18 as can their processing by the RNase III enzyme Drosha,19 and their stability also can be highly regulated.20,21 The molecular mechanisms controlling microRNA processing and stability are only beginning to be unraveled. Considering the large number of mRNAs that may be regulated by each microRNA, the dysregulation of microRNA expression therefore can have profound effects on multiple cellular processes. Pioneering work in Caenorhabditis elegans (C elegans) on the first microRNA ever studied, lin-4, revealed that microRNAs can affect developmental cellfate decisions; in this case through the regulation of the heterochronic genes, lin-14 and lin-28.22,23 The phenotype of mice deficient in Dicer, an enzyme required for microRNA biogenesis, has emphasized that microRNAs are key regulators of mammalian development because loss of Dicer leads to embryonic lethality around embryonic day 7.5.24 Mice homozygous for a hypomorphic allele of Dicer also die during embryogenesis (embryonic days 12.5-14.5), apparently from defects in blood vessel development.25 In addition to crucial developmental roles, microRNAs also can regulate postnatal physiology. Surprisingly, endothelial-specific deletion of Dicer does not alter vascular development, but postnatal defects are observed in the response of endothelial cells to angiogenic factors, and the growth of new blood vessels in models of hind limb ischemia, wound healing, and tumorigenesis.26 Several groups also have reported that the deletion of Dicer in specific cell types within the kidney of mice does not result in obvious developmental abnormalities, but instead results in defects in postnatal kidney function. For instance, deletion of Dicer in juxtaglomerular cells results in failure to maintain this cell type, leading to defective renin production, lower blood pressure, and arteriolar defects and fibrosis in the kidney.27 In addition, Dicer deletion in podocytes results in cytoskeletal defects, apoptosis, proteinuria, foot process effacement, and

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defects in the glomerular basement membrane.28-30 This phenotype is evident 2 to 4 weeks after birth, and eventually leads to glomerulosclerosis, tubulointerstitial fibrosis, and subsequent kidney failure by 6 to 8 weeks of age. In contrast to the rather severe postnatal phenotype of Dicer deletion in podocytes or juxtaglomerular cells, conditional ablation of Dicer in proximal tubule cells fails to induce a postnatal phenotype under normal cellular conditions. However, these mice are remarkably protected from renal ischemia-reperfusion injury.31 There is decreased tissue damage, less tubular cell apoptosis, and improved kidney function and overall survival in tubule-specific Dicer -/- mice. Taken together, these Dicer loss-of-function experiments underscore the fact that microRNAs function in various aspects of cell biology, but because the expression of many microRNAs are lost in these Dicer mutants, it is difficult to identify the pathways that each microRNA regulates using this sledgehammer approach. Not surprisingly, loss-of-function of individual microRNAs often results in more subtle phenotypes than total loss-of-function of microRNAs via Dicer deletion. In fact, the majority of single microRNA knock-outs generated in C elegans do not display overt defects.32 This suggests that although microRNAs collectively may control a plethora of cellular pathways, loss of a single microRNA often is tolerated, perhaps because microRNAs fine-tune gene expression, rather than acting as potent on/off switches. A seminal finding with respect to dissecting the function of a single microRNA in vivo came from work characterizing the phenotype of one of the first genetic deletions of a microRNA in the mouse. Mice deficient in the muscle-specific microRNA, miR-208, which is embedded in an intron of ␣-myosin heavy chain, appear normal during development and are viable and apparently healthy.33 However, these mice are unable to initiate the normal response to cardiac stress or hypothyroidism. Indeed, this microRNA is required for stress-induced fibrosis, hypertrophy, and expression of the fetal gene program, including expression of ␤-myosin heavy chain.33 A similar role for microRNAs in the control of the cellular stress response was identified in mice deficient in the vascular smooth muscle– enriched microRNA cluster, miR-143/145.2,4 Genetic deletion of these microRNAs does not significantly affect the development of blood vessels, but neointimal formation after vascular injury is attenuated. These findings suggest that microRNAs can function as homeostatic regulators by controlling the cellular response to injury or stress. Phenotypic Diversity of the Endothelium and Vascular Disease Although rapid proliferation and migration of endothelial cells is required for the formation of the vascular network during development, endothelial cells acquire a quiescent phenotype in the vasculature of the adult. Embryonic endothelial cells and adult endothelial cells are illustra-

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tive of the range of endothelial phenotypes that are possible. Indeed, among endothelial cells in different organ systems in the adult, and even in unique vascular beds within the same organ or tissue, endothelial cells are dramatically heterogenous in their morphology, gene expression profiles, and function (reviewed by Aird34). In the kidney, for example, fenestrated endothelial cells are located in the glomerulus and in a subset of the renal tubules, whereas continuous endothelial cells line the majority of the other vessels in the kidney. Fenestrated endothelial cells contain large pores (60-80 nm) and play a specialized role in filtration, clearly an essential function of the kidney. This specialized phenotype can be contrasted with the endothelium that constitutes the blood-brain barrier, which lack fenestrations and have numerous tight junctions to achieve restricted vascular permeability. This structural and functional heterogeneity among endothelial cells not only facilitates specialized vascular functions, but it also has a well-appreciated role in the regional susceptibility to vascular disease. Although some aspects of endothelial phenotype may be locked in by epigenetic mechanisms,35,36 other phenotypic properties are actively maintained by the cellular microenvironment.37 Dynamic phenotypic alterations can occur because endothelial cells are constantly sensing and responding to their environment. For example, local ischemia can promote the loss of endothelial quiescence and cell– cell junctions and the initiation of an angiogenic program that promotes the proliferation and migration of endothelial cells, resulting in the formation of a new blood vessel to provide oxygen to the ischemic area.38 Interestingly, it has been suggested that vascular diseases associated with aging, such as atherosclerosis, may result from a decrease in the ability of endothelial cells to dynamically modulate vascular function in a contextappropriate manner; including maintaining vascular tone and the antithrombotic and anti-inflammatory surface of

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the vessels, and controlling the response to mitogenic stimuli.39 Aging also affects the function of circulating endothelial progenitor cells (EPCs), which participate in vascular repair. EPCs can become senescent during aging or disease, which influences the number of these cells in the circulation, their migratory capacity, their sensitivity to apoptosis, and their ability to mediate repair (reviewed by Herrera et al39). Although the ability of endothelial cells to adapt to changing environments is key to the maintenance of homeostasis, chronic changes in cellular phenotype in response to these stimuli can lead to disease. For example, regional differences in the physical forces of the circulation are known to dramatically affect endothelial phenotype.40,41 This flow-based modulation of endothelial phenotype influences the ability of the vasculature to respond to environmental cues, such as inflammatory cytokines, and particular regions of the vascular tree consequently have a heightened susceptibility to the development of disease.42-44 Considering the importance of endothelial phenotype to the maintenance of cardiovascular homeostasis and the involvement of phenotypic changes in the etiology of vascular disease, endothelial cells must tightly regulate how they respond to stimuli in their environment. Recent work is uncovering novel roles for microRNAs in regulating this process. microRNA Regulation of Vascular Function: Angiogenesis Recently, several microRNAs were identified as key regulators of angiogenic signaling pathways in endothelial cells (reviewed by Fish and Srivastava45). For example, miR-126 promotes angiogenesis by repressing negative regulators of angiogenic signaling pathways46-49 (Fig. 1), and this microRNA promotes vessel growth in hindlimb ischemic models,50 as well as after myocardial infarction.49 The host gene of miR-126, Egfl7, is expressed at low levels in quiescent endothelium, but is up-regulated

Figure 1. miR-126 negatively regulates angiogenic signaling. (A) Schematic of the targeting of Sprouty-related, EVH1 domain-containing 1 (SPRED1) and phosphoinositide 3-kinase, regulatory subunit 2 (␤) (PIK3R2), negative regulators of angiogenic signaling, by the endothelialspecific microRNA, miR-126. PI3K, phosphoinositide 3-kinase pathway; MAPK, mitogen-activated protein kinase pathway. (B) In contrast to control cells, miR-126 knock-down endothelial cells fail to initiate an angiogenic program in response to Vegf stimulation, as observed by their reduced rate of migration into the scratch wound. Shown is phalloidin staining, which labels the actin cytoskeleton.

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in tumor endothelium,51 suggesting that miR-126 also might be up-regulated in the setting of pathologic angiogenesis, and therefore would be a useful and specific target for cancer therapies. Anti-angiogenic microRNAs also have been identified, and their effect on tissue regeneration has been investigated. For example, miR-92a negatively regulates angiogenic responses, in part by targeting the pro-angiogenic factor, integrin ␣5. Inhibiting the expression of this microRNA enhances vascular growth after hindlimb ischemia or myocardial infarction.52 Other members of the miR-17-92 microRNA cluster, including miR-17 and miR-20a, also repress angiogenic processes.53 We recently found that miR-218, which is processed from an intron of Slit-2 and Slit-3, negatively regulates angiogenic signaling by repressing the Slit receptor, Robo-1.54 Robo-1 appears to potentiate the vascular endothelial growth factor (Vegf) signaling pathway,54 and knock-down of miR-218 has been shown to affect the patterning of the retinal vasculature.55 Additional examples of microRNAs that modulate angiogenesis are miR-130a, which targets the anti-angiogenic homeobox genes, Hoxa5 and Gax56; miR-221/222, which targets c-Kit57; and let-7f, which promotes angiogenesis by targeting thrombospondin-1.58 In addition, miR-296 is up-regulated by angiogenic factors and facilitates angiogenesis by repressing hepatocyte growth factor–regulated tyrosine kinase substrate, which degrades Vegf receptor 2 and platelet-derived growth factor-␤.59 Seminal work directed at elucidating the pathways involved in renal cancer etiology revealed that von Hippel Lindau protein, which commonly is mutated in renal clear cell carcinoma, controls hypoxic signaling by promoting the degradation of hypoxia inducible factors.60 Hypoxia is a potent inducer of angiogenesis during development and in adult vascular pathogenesis. This is partly owing to the induction of angiogenic factors such as Vegf by hypoxia inducible factor–mediated transcription. Recent work has highlighted the effect that hypoxia has on microRNA expression. In the endothelium, hypoxia induces the expression of miR-210, which promotes hypoxia-dependent capillary sprout formation by repressing Ephrin-A3.61 This microRNA also regulates endothelial mitochondrial metabolism under hypoxic conditions.62 Several groups have observed hypoxia-dependent microRNA regulation in tumor cells. This group of microRNAs has been referred to as hypoxamirs, and the expression of a particular signature of hypoxia-regulated microRNAs is found in many human tumors.63,64 In addition to hypoxia, oncogene activation in tumor cells promotes tumorigenesis in a microRNA-dependent manner. For example, expression of the miR-17-92 cluster is regulated by the Myc oncogene, and this microRNA family represses the anti-angiogenic molecules thrombospondin-1 and connective tissue growth factor, which in turn promotes angiogenesis.65 These findings highlight the importance of hypoxia- and oncogene-regulated

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microRNAs in controlling tumorigenesis via regulation of factors that affect the activation of the endothelium. The activity of these angiogenesis-regulating microRNAs might be harnessed or repressed to control vascular growth in the context of solid tumors or in the setting of ischemic insults such as myocardial infarction. microRNA Regulation of Vascular Function: Endothelial Cell Senescence Although the activation and proliferation of endothelial cells within blood vessels can aid in the response to tissue injury, it is now appreciated that circulating cell types also can contribute to repair. For example, bone marrow– derived EPCs can home to sites of injury,66 where they can either secrete angiogenic mitogens or directly differentiate into new endothelium. The contribution of EPCs to tissue repair and pathologic processes such as tumor angiogenesis or atherosclerosis is intriguing but poorly understood (reviewed by Liu et al67). Interestingly, atherosclerosis has been suggested to be a chronic inflammatory disease that may result from a failure to repair damaged endothelial cells after cellular injury, implying that repair of damaged endothelium is an important facet of this disease. The finding that the number of EPCs in patient samples is correlated negatively with known risk factors for vascular disease suggests that these cells may be important in the host response to injury.68 Not surprisingly, the replicative potential of EPCs determines their ability to participate in vascular repair. A growing body of research has studied the factors that affect endothelial senescence, and in particular how this phenomenon regulates EPC viability and utility (reviewed by Herrera et al39). Interestingly, miR-34a is expressed in EPCs and increased levels of this microRNA can promote endothelial senescence through negative regulation of silent information regulator 1 (SIRT1).69 MicroRNA-based regulation during senescence also may be relevant to vascular endothelial cells. MiR-217 is up-regulated in senescent endothelial cells and this microRNA also targets SIRT1.70 Decreased SIRT1 results in less Forkhead box O1 (FoxO1) and endothelial nitric oxide synthase acetylation and a consequent reduction in angiogenesis. Conversely, inhibition of miR-217 enhances the angiogenic properties of senescent endothelial cells. Importantly, miR-217 is expressed in atherosclerotic lesions and its expression is correlated inversely with the acetylation status of FoxO1 and endothelial nitric oxide synthase.70 These findings suggest a key role for microRNAs in controlling tissue repair, including the regulation of angiogenic processes and cellular senescence pathways. An understanding of the mechanisms used by these microRNAs, including the pathways targeted, will facilitate therapeutic targeting in the setting of tissue injury and disease. Whether these concepts are relevant to diseases of the kidney, especially glomerular injury, will require future studies.

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microRNA Regulation of Vascular Function: Inflammation The innate immune system defends against invading viruses and other foreign pathogens. Monocytes and other immune cells respond to viruses or bacterial cell components through the activation of Toll-like receptor (TLR) pathways that culminate in the expression and secretion of proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (reviewed by Barton and Medzhitov71). In addition to their pyrogenic effects, these cytokines act on the endothelium to promote blood vessel permeability and the induction of adhesion molecule expression at the site of inflammation; both of which facilitate immune cell recruitment and activity. These cytokines also facilitate endothelial dysfunction, including the loss of vascular tone and a failure to inhibit thrombosis. Activation of the innate immune system also plays a critical role in the pathogenesis of atherosclerosis. Monocytes are recruited to endothelial cells expressing adhesion molecules such as vascular cell adhesion molecule-1, and then migrate into the vessel wall, where they can differentiate into macrophages and foam cells, which produce high levels of inflammatory cytokines that mediate further endothelial dysfunction and promote plaque instability (reviewed by Tedgui72). Recently, it was discovered that microRNAs are induced by the activation of the innate immune response. An emerging theme from several studies is that these microRNAs form a negative feedback loop to quench TLR signaling. An example of such a feedback system involves miR-146, which is induced by lipopolysaccharide and targets TNF-receptor–associated factor 6 (TRAF6) and interleukin-1–receptor–associated kinase 1 (IRAK1), two components of the TLR pathway73 (Fig. 2). This negative feedback loop may contribute, at least in part, to the phenomenon of endotoxin-induced tolerance.74 Similar roles also have been described for miR147 and miR-155, which also are induced by TLR signaling and negatively regulate inflammatory cytokine production75,76 (Fig. 2). An analogous negative feedback loop also appears to function in endothelial cells to modulate inflammatory signaling pathways. Treatment of endothelial cells with TNF-␣ up-regulates miR-31 and miR-17-5p, which in turn target the adhesion molecules E-selectin and intercellular adhesion molecule-1, respectively77 (Fig. 2). The up-regulation of these microRNAs inhibits neutrophil adhesion to endothelial cells, a key step in the pathogenesis of atherosclerosis. Perhaps this microRNA-based feedback regulation of inflammatory signaling tempers the inflammatory response to prevent the potentially devastating consequences of an overactivation of this pathway. In support of this hypothesis, microRNAs have been found to be dysregulated in autoimmune diseases (reviewed by O’Connell et al78). One of the most highly expressed endothelial microRNAs is miR-126. Although miR-126 levels do not appear to be dynamically regulated by inflammatory signaling path-

Figure 2. MicroRNAs regulate innate immunity and the inflammatory response. MiR-146, miR-147, and miR-155 are activated in immune cells in response to activation of the innate immune pathway. These microRNAs participate in a negative feedback loop to temper the immune response. Whether these microRNAs are induced in endothelial cells in response to lipopolysaccharide (LPS) is not known. Also shown is the regulation of vascular cell adhesion molecule-1 (VCAM1) expression by the endothelial-specific microRNA, miR-126. Treatment of endothelial cells with the proinflammatory cytokine, TNF-␣, results in the induction of miR-31 and miR-17-5p, which act to limit the inflammatory response by targeting intercellular adhesion molecule 1 (ICAM1) and E-selectin (SELE), respectively. TNFR, tumor necrosis factor receptor; IRAK1, interleukin-1-receptor-associated kinase 1; TRAF6, TNF-receptor-associated factor 6.

ways,77 this microRNA can negatively regulate the expression of the inflammatory adhesion molecule, vascular cell adhesion molecule-1 (VCAM1)46,79 (Fig. 2). MiR126 also is enriched in apoptotic bodies that are generated from dying endothelial cells during the progression of atherosclerosis, and this source of miR-126 can repress regulator of G-protein signaling 16, a negative regulator of stromal-derived factor 1 signaling, thereby inhibiting atherosclerosis progression and enhancing plaque stability.80 Therefore, in addition to its role in regulating angiogenic signaling cascades, miR-126 also appears to function as an anti-inflammatory mediator in endothelial cells. Flow dynamics play a crucial role in regulating blood vessel biology, and regional differences in these dynamics regulate inflammation and the progression of atherosclerosis. In particular, areas of the vasculature that are exposed to high levels of laminar shear stress are quiescent and protected against inflammation and the development of atherosclerosis, whereas regions of disturbed flow, which typically is found at branch points and the inner curvature of arched vessels, have increased endothelial turnover and are prone to inflammation and atherosclerosis (reviewed by VanderLaan et al81). Endothelial cells initiate a shear stress– dependent gene expression program that includes modulation of microRNA expression. For example, miR-19a is induced after 12 hours of laminar flow and negatively regulates cyclin D1 expression to control

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endothelial proliferation.82 In addition, pulsatile flow patterns up-regulate miR-23b and miR-27b, which promote G0/G1 growth arrest.83 Finally, miR-21 also is induced by laminar flow and represses phosphatase and tensin homolog (PTEN), inhibits apoptosis, and promotes endothelial nitric oxide synthase activity.84 The same physical forces of the circulation that govern pathology of the adult vasculature are likely also important during development of the vascular network during embryogenesis. Recently, studies in zebrafish have identified a role for flow dynamics in the regulation of miR126 and the control of branchial arch remodeling.48 In zebrafish, miR-126 is induced by shear stress and targets spred1, a negative regulator of angiogenic signaling, to control responses of endothelial cells to Vegf. Whether flow-dependent regulation of microRNAs controls the growth of the vascular network during mammalian development has yet to be explored. Considering that the expression of several microRNAs are dynamically regulated in response to inflammatory signals and/or altered hemodynamics, it is likely that microRNA function contributes to the development of atherosclerosis, which has many features of a chronic inflammatory disease. Several studies have begun to investigate the role that microRNAs play in the development and progression of atherosclerosis, although to date no studies have focused on microRNAs in the vascular endothelium. In models of neointimal formation the expression of several microRNAs becomes dysregulated. Inhibition of miR-21 in vivo, which is induced under these conditions, inhibits smooth muscle cell proliferation and neointimal formation.7 Of note, miR-21 also is highly expressed in the endothelium,58 but the role of miR-21 in this cell type has not been addressed directly. Other studies have addressed the effect of oxidized lowdensity lipoprotein on inflammatory mediators. miR125a-5p is up-regulated by oxidized low-density lipoprotein treatment of monocytes and endothelial cells and negatively regulates the expression of proinflammatory cytokines,85 and also targets the vasoconstrictor, endothelin-1,86 again emphasizing the importance of microRNAs to negative feedback regulatory loops. In addition, miR-221 and miR-222 levels are increased in EPCs isolated from patients with coronary artery disease, but levels are reduced in EPCs isolated from patients receiving long-term statin therapy. Importantly, EPC numbers correlated negatively with the level of miR-221/222.87 The mechanistic basis for this correlation is not known. Taken together, these preliminary studies suggest that microRNAs might importantly contribute to atherosclerosis.

FUTURE DIRECTIONS The endothelium does not simply act as a barrier between the blood and the rest of the body, but plays an active role in maintaining blood vessel homeostasis. To achieve this, the endothelium dynamically responds to alterations in

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the cellular microenvironment. It is now clear that microRNAs play a role in controlling several aspects of endothelial biology, including angiogenic and inflammatory signaling, and senescence pathways. Understanding the contribution of microRNAs to vascular disease is a burgeoning field. As the field continues to mature from simple cataloging of the microRNAs affected by stimuli to an in-depth understanding of the targets involved and the cellular pathways regulated by these microRNAs, we will gain a more comprehensive understanding of the contribution of these small RNAs to vascular disease. One limitation that continues to slow advances in this field is the lack of highly accurate prediction programs to identify the targets of microRNAs. It will be especially important to determine the physiologically relevant targets of microRNAs because many studies to date have relied on overexpression of microRNAs and/or use heterologous systems such as luciferase assays to identify microRNA targets. Further complicating target prediction, microRNAs also can bind and repress mRNAs in a seed-independent manner.14 New biochemical methods for target identification88 will allow for a better understanding of the rules that govern microRNA/target interactions, and the use of these methods in relevant cell types will enhance our understanding of microRNA biology. Recent work has shown that microRNAs can be detected in patient blood samples,89 and there are some data that suggest that microRNAs even may be transferred between distal cells by exosomes.90,91 MicroRNAs therefore may be promising biomarkers for a number of disease states, including cancer and cardiovascular disease. Intriguing findings also have been made with respect to the presence of single nucleotide polymorphisms in microRNA binding sites within the 3’ UTRs of target mRNAs92 and within microRNA genes.93 These single nucleotide polymorphisms may have the potential to contribute to the genetic predisposition to various diseases. Advances in the use of microRNAs as therapeutics also are encouraging. For example, technology to antagonize microRNA function through the use of antisense inhibitors is improving with respect to the stability of these inhibitors and their tissue distribution, and these inhibitors now have been used successfully in primate models to lower cholesterol by targeting the liver-specific microRNA, miR-122.94 Because of limitations in our ability to inhibit microRNA activity in one particular tissue or organ system, microRNAs with cell-restricted expression patterns, such as the liver-specific miR-122,95 or the endothelial-specific miR-126,46,49 initially will be more attractive therapeutic targets, but novel methods of targeting microRNA mimetics or inhibitors to particular regions of the body (in particular, regions of ischemia or tumor growth), will broaden their therapeutic potential. We have just begun to understand the various contributions of microRNA-based regulation of disease process, and the progress in this area undoubtedly will provide a

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more comprehensive view of how to detect and treat human diseases of the vasculature and are highly relevant to the kidney.

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ACKNOWLEDGEMENTS I thank Dr. Joshua Wythe (Gladstone Institute of Cardiovascular Disease, San Francisco, CA) for critical reading of this manuscript.

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