Role for miR-181 family in regulating vascular inflammation and immunity

Abstract

The microRNA family, miR-181, plays diverse roles in regulating key aspects of cellular growth, development, and activation. Accumulating evidence supports a central role for the miR-181 family in vascular inflammation by controlling critical signaling pathways, such as downstream NF-κB signaling, and targets relevant to endothelial cell activation and immune cell homeostasis. This review examines the current knowledge of the miR-181 family’s role in key cell types that critically control cardiovascular inflammation under pathological and physiological stimuli.

Introduction

Vascular inflammation occurs as a consequence of infection, metabolic stress, autoimmune response, and other intrinsic or extrinsic insults to the blood vessel wall. The cellular response typically involves not only endothelial cells (ECs), but also a range of leukocytes such as monocytes/macrophages, dendritic cells, lymphocytes, and neutrophils. Under physiological conditions, the vascular endothelium confers protective mechanisms against inflammation including the maintenance of blood fluidity, control of vessel wall permeability, and quiescence of circulating leukocytes (Pober and Sessa, 2007). ECs are induced to express adhesion molecules and produce inflammatory cytokines by diverse inflammatory stimuli which act in an autocrine and paracrine manner to fuel the inflammatory response. The activated endothelium, in turn, creates a pro-inflammatory environment to support leukocyte recruitment toward inflamed sites. Leukocytes are key players in vascular inflammation (Moore and Tabas, 2011; Weber et al., 2008). For example, in response to stimuli, monocytes/macrophages generate a wide array of biologically active products including cytokines and chemokines that further propagate the initial stimulus. Macrophages, phagocytic cells by nature, engulf debris from damaged host cells and pathogens. In both ECs and leukocytes, NF-κB signaling is a central pathway mediating the pathogenesis of acute (e.g. sepsis) and chronic inflammatory disease states (e.g. atherosclerosis, diabetes, rheumatoid arthritis, inflammatory bowel disease). In acute vascular inflammation, inflammatory responses are typically tightly controlled and eventually resolve. Unresolved vascular inflammation can contribute to chronic inflammatory diseases such as atherosclerosis (Baker et al., 2011; Dutta et al., 2012; Libby, 2002, 2012; Libby et al., 2011).

MicroRNAs (miRNAs), small non-coding single-stranded RNA molecules, have emerged as key regulators of gene expression at the post-transcriptional level by inhibiting mRNA translation and/or promoting mRNA degradation. MiRNAs play crucial roles in various physiological and pathological processes such as immune cell differentiation, EC activation, and various aspects of vascular inflammation (Urbich et al., 2008; Weber et al., 2010; Wei et al., 2013). In this review, we summarize the emerging roles of miR-181 family members and their targets in EC biology, leukocyte biology, and vascular inflammation (Table.1).

Table 1.

Targets of miR-181 family members involved in vascular biology and immunity

miRNA	Targets	Functions	References
miR-181a	Osteopontin	Regulates osteopontin-mediated VSMC function	Remus et al. (2013)
	Dusp6	Decline in miR-181a expression with age impairs T cell receptor sensitivity	Li et al. (2012)
	c-Fos	Represses ox-LDL-stimulated inflammatory response in dendritic cell	Wu et al. (2012)
	Prox1	Control of Prox1 expression during vascular development and neo-lymphangiogenesis	Kazenwadel et al. (2010)
	unknown	Regulates thymic positive selection, and enforces central tolerance	Ebert et al. (2009)
	Shp-2, Ptpn22, DUSP6	Acts as an intrinsic modulator of T cell sensitivity and selection	Li et al. (2007)
	Bcl-2, CD69, TCR	Involved in T cell maturation and positive selection	Neilson et al. (2007)
	unknown	Modulates hematopoietic lineage (B-lineage cells) differentiation	Chen et al. (2004)
miR-181b	Importin-α3	Inhibits downstream canonical NF-κB signaling pathway in endothelial cells	Sun et al. (2012)
		Reduces vascular inflammation in vivo
		Decreases lung inflammation in endotoxemia mice
		Promotes survival in LPS-induced septic mice
		Expression is reduced in septic patients and in endotoxemic mice
	NRP1	Reduction of miR-181b mediates arsenic-induced angiogenesis	Cui et al. (2012)
	AID	Regulates AID-dependent reactions in activated B cells	de Yébenes et al. (2008)
miR-181c	BMPR2	May be associated with the pathogenesis of ventricular septal defects	Li et al. (2013)
	mt-COX1	Regulates mitochondrial genome expression and function	Das et al. (2012)
	IL-2	Serves as a negative regulator that modulates the activation of CD4(+) T cells	Xue et al. (2011)
miR-181a/b	PTEN	Controls PI3K signaling, cell anabolic metabolism, and NKT cell development	Henao-Mejia et al. (2013)
	Ptpn22, Shp-2, and Dusp6	Sets a TCR signaling threshold, promotes invariant natural killer T (iNKT) cell development	Zietara et al. (2013)
miR-181d	LIF	Regulates the acute stress response in thymocytes	Belkaya et al. (2011)

Dusp6: dual specificity phosphatase 6

Prox1: prospero homeobox 1

Shp-2: SH2-containing protein tyrosine phosphatase-2

Ptpn22: protein tyrosine phosphatase nonreceptor type 22

Bcl-2: B-cell lymphoma/leukemia 2

CD69: Cluster of Differentiation 69

TCR: T cell receptor

NRP1: neuropilin-1

AID: activation-induced cytidine deaminase

BMPR2: Bone morphogenetic protein receptor type II

mt-COX1: mitochondrially encoded cytochrome c oxidase I

PTEN: Phosphatase and tensin homolog

LIF: leukemia inhibitory factor

Genomic location of miR-181 family members

More than 2,000 mature miRNAs exist in the human genome, and the list of miRNAs is continuously growing (http://www.mirbase.org/). MiRNAs are dispersed throughout the genome often found between independent transcription units (intergenic), or more commonly in the intronic sequences of protein-coding genes and intronic/exonic regions of noncoding RNAs (intronic) (Rodriguez et al., 2004; Saini et al., 2007). Intergenic miRNAs genes have their own promoters and terminators, while the majority of intronic miRNAs share the same transcription elements with their host genes. The human and mouse miR-181 family constitutes four members (miR-181a, miR-181b, miR-181c, and miR-181d). They are encoded by three different transcripts located on three different chromosomes (Figure.1A). MiR-181a and miR-181b are well-studied members of the miR-181 family and cluster together on two genomic locations: the human miR-181-a1 and miR-181-b1 cluster is located on chromosome 1; the miR-181a2 and miR-181b2 cluster is located on chromosome 9. The miR-181c and miR-181d cluster is located on chromosome 19. These miR-181 family members contain similar seed sequences that may differ in one to four nucleotides only (Figure. 1B). For instance, mature miR-181a and miR-181c sequences, or miR-181b and miR-181d sequences, have only one nucleotide difference. When two mature miRNAs are generated from the opposite arms of the same miRNA precursor, the mature miRNAs that arise from the 5′ or 3′ arm of the precursor are denoted with a -5p or -3p suffix, respectively. Human miR-181a1, miR-181b1, miR-181a2, and miR-181c encode both -5p and -3p mature miRNAs, whereas those generated from the 3′ arms are listed in Figure 1C. Whether both -5p and -3p miR-181 members have similar biological functions has not been examined. Since -5p and -3p miR-181s have different seed sequences, they likely target different genes and pathways. Finally, although -5p miR-181 family members have the same seed sequence, they have distinct gene targets. For example leukemia inhibitory factor was targeted by miR-181d but not miR-181a (Belkaya et al., 2011). In addition, while miR-181a and miR-181c encode almost identical mature miRNAs, the strength and functional specificity of these miRNAs may be modulated through their pre-miRNA loop nucleotides (Liu et al., 2008). MiR-181a1, but not miR-181c, can promote CD4 and CD8 double-positive T cell development which is largely determined by their unique pre-miRNA loop nucleotides (Liu et al., 2008). This highlights the non-redundant role of miR-181 family members, and adds a potential new layer of complexity for their role in cardiovascular inflammation.

Figure 1.

MiR-181 family members and their genomic locations. A, genomic location of miR-181 family members. B, mature sequences of miR-181 family members that arise from the 5′ arm of the precursors. Nucleotides in red represent seed sequence, and green shows conserved nucleotides among the members. C, mature sequences of miR-181 family members that arise from the 3′ arm of the precursors.

Role of miR-181 family members in endothelial cells

EC activation and dysfunction is involved in the pathogenesis of many diseases such as atherosclerosis, rheumatoid arthritis, and diabetes. Perturbations in EC function are associated with the activation of signaling pathways and changes in gene expression. The role of several miRNAs in controlling EC function and vascular homeostasis has been reviewed elsewhere (Boon et al., 2012; Chamorro-Jorganes et al., 2013; Fish, 2012; Hartmann and Thum, 2011). In this section, we will focus on the role of miR-181 family members in controlling EC specification, function, and vascular inflammation.

One of the important components of the cardiovascular system is the lymphatic vasculature which facilitates returning interstitial fluid and proteins to the bloodstream, acts as a conduit for migration of immune cells during inflammation, and helps to absorb lipoproteins from the gut. Thus, it is critical that the specification of lymphatic EC (LEC) fate is tightly and precisely controlled. The homeobox transcription factor prospero homeobox 1 (Prox1) is recognized as a master regulator of LEC identity and function. Overexpression of miR-181a reduced Prox1 expression by binding to its 3′-untranslated region (3′-UTR) (Kazenwadel et al., 2010). The expression of miR-181a and Prox1 are reciprocal in different tissues during development. The higher expression of miR-181a expression observed in embryonic blood vascular ECs compared to lymphatic vascular ECs may therefore help to prevent of the acquisition of LEC identity in blood vascular endothelial cells by silencing Prox1 expression in these cells (Kazenwadel et al., 2010).

The activated endothelium, whether activated by biochemical or biomechanical stimuli, contributes to the initiation and progression of vascular inflammatory disease states. Sepsis is a medical condition that represents a patient’s systemic inflammatory response to a severe infection (Iskander et al., 2013). Endotoxin lipopolysaccharide (LPS) is involved in the pathogenesis of about 25%–30% sepsis cases (Annane et al., 2005), which activates a Toll-like receptor (TLR)-mediated signaling pathway and NF-κB, induces the release of critical pro-inflammatory cytokines including (Tumor necrosis factor-alpha) TNF-α, activates the vascular endothelium, and elicits a systemic inflammatory response syndrome (Aird, 2003; Lu et al., 2008; Nduka and Parrillo, 2009). We recently reported that miR-181b serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium, and found that this effect is mediated by direct targeting of importin-α3, a protein critical for NF-κB translocation from the cytoplasm to the nucleus (Sun et al., 2012). Using microarray miRNA profiling, miR-181b was identified as rapidly reduced in response to TNF-α stimulation in ECs. Furthermore, MiR-181b expression is reduced in the vascular endothelium of mice in response to pro-inflammatory stimuli such as TNF-α or LPS. Overexpression of miR-181b suppressed an enriched set of NF-κB-responsive genes in ECs, such as adhesion molecules VCAM-1 and E-selectin, and reduced leukocyte adhesion to activated EC monolayers. Systemic administration of miR-181b ‘mimics’ reduced p65 nuclear accumulation in the vascular endothelium, leukocyte accumulation, lung inflammation, and improved survival by ~50% in endotoxemic mice. In contrast, systemic delivery of miR-181b inhibitors potentiated LPS-induced expression of NF-κB-responsive genes such as VCAM-1 and E-selectin, NF-κB signaling, and lung injury in vivo. Interestingly, the reduced expression levels of miR-181b in endotoxemic mice may have clinical relevance as we observed reduced levels of circulating miR-181b expression in critically ill patients with sepsis compared to control subjects in the intensive care unit.

We identified that miR-181b inhibits the activation of NF-κB signaling by targeting the 3′-UTR of the gene which encodes importin-α3. This target was identified after a series of studies that led to the surprising observation that miR-181b inhibited downstream, and not upstream, NF-κB signaling. We first appreciated that miR-181b inhibited transcriptional NF-κB and VCAM-1 promoter activity. Second, miR-181b inhibited p65 nuclear expression by confocal microscopy. Finally, separation of EC nuclear and cytoplasmic cell extracts revealed that while miR-181b reduced p65 and p50 expression in the nuclear fraction (and increased expression in the cytoplasmic fraction), there was no effect of miR-181b on upstream effectors including the IKK complex, IκBα, or IκBα-phosphorylation. The latter finding raised the possibility that miR-181b may inhibit downstream NF-κB signaling by targeting the NF-κB translocation step. Accumulating studies highlight NF-κB translocation from cytoplasm-to-nucleus is not a passive process, but an active one, that requires a family of proteins termed importins(Fagerlund et al., 2005; Fagerlund et al., 2008; Kohler et al., 1999; Theiss et al., 2009). These studies highlight that several importin-α members (in particular, importin-α3, importin-α4, and importin-α5) heterodimerize to NF-κB family members such as p65 and p50 (Fagerlund et al., 2005; Fagerlund et al., 2008). Indeed, using in silico prediction algorithms, we identified several potential importin-α members as targets; however, only importin-α3 expression and its 3′-UTR was uniquely repressed by miR-181b in ECs (Sun et al., 2012).

Several lines of evidence further substantiated that importin-α3 is a bone fide target of miR-181b in ECs: 1) mutation of 2 consensus miR-181 binding sites blocked miR-181b-mediated repression of importin-α3; 2) Argonaute2-immunoprecipitation studies revealed that miR-181b associated directly with importin-α3 mRNA which was enriched 5-fold compared to a non-specific control miRNA mimic; 3) overexpression of importin-α3 lacking its 3′-UTR could effectively rescue the miR-181b-mediated inhibition of NF-κB activity and target genes (e.g. VCAM-1, E-selectin) in vitro and in lung ECs in vivo; 4) siRNA-mediated ‘knockdown’ of importin-α3 expression recapitulated the inhibitory effects of miR-181b on NF-κB in ECs in vitro and in vivo; and 5) in the presence of siRNA-mediated knockdown of importin-α3 in vivo, miR-181b’s inhibitory effects on NF-κB was significantly blocked (Sun et al., 2012). Moreover, the inhibitory effect of miR-181b was selective for the NF-κB signaling pathway as miR-181b had no effect on major MAPK downstream signaling pathways including ERK1/2, p38, or JNK. Importantly, miR-181b’s inhibitory effect was specific to ECs as no reduction in NF-κB activity was observed in peripheral blood mononuclear cells in endotoxemic mice in vivo. Collectively, these findings implicate miR-181b as a novel regulator of NF-κB-mediated EC activation and vascular inflammation in response to pro-inflammatory stimuli and substantiate that restoration of miR-181b expression in ECs may provide a new strategy for anti-inflammatory therapy (Figure 2). Whether other miR-181 family members have similar protective, inhibitory effects as miR-181b on NF-κB signaling, EC activation, and vascular inflammation and sepsis will require further investigation.

Figure 2.

MiR-181b inhibits NF-κB-mediated vascular inflammation by targeting importin-α3 in endothelial cells.

Arsenic is a well-known naturally occurring toxic element that has serious health effects on humans. The underlying pathogenesis of arsenic-induced toxicity and diseases is associated with EC activation and dysfunction (Balakumar and Kaur, 2009; Wang et al., 2012). A set of miRNAs including miR-181b exhibited reduced expression in chick embryos after arsenite administration. Neuropilin-1 (NRP1) is a single spanning transmembrane glycoprotein, which plays versatile roles in angiogenesis, axon guidance, cell survival, and migration. MiR-181b suppressed NRP1 expression through a specific 3′-UTR binding site. MiR-181b overexpression or NRP1 knockdown inhibited EC migration and tube formation in the presence of arsenite. These data suggest the reduction of miR-181b after arsenite exposure at least partially mediates arsenite-induced EC migration and angiogenesis because of the increase of NRP1 expression (Cui et al., 2012).

Role of miR-181 family members in fibroblasts, cardiomyocytes, and vascular smooth muscle cells

In response to cardiac injury, both cardiomyocytes and fibroblasts become susceptible to a range of stimuli such as reactive oxygen species and angiotensin II (Ang II) that may confer deleterious effects on cellular apoptosis, necrosis, and remodeling, among other processes (Dostal et al., 1997; Kajstura et al., 1997; Matsusaka et al., 1999; Souders et al., 2009; Sun, 2007; Zhang et al., 2001). In response to Ang II, miR-181b expression was markedly decreased by 3.5-fold in cardiac fibroblasts suggesting its potential role in cardiac fibrosis and hypertension (Jiang et al., 2013). In cardiomyocytes, miR-181c can translocate into mitochondria to regulate mitochondrial function (Das et al., 2012). Remarkably, in mitochondrial cell fractions, miR-181c targeted mitochondrial cytochrome c oxidase subunit 1 (mt-COX1) expression as verified by interaction with its mRNA after argonaute-2 immunoprecipitation and by inhibition of the mt-COX1 3′-UTR activity suggesting mt-COX1 is a direct target of miR-181c in cardiac myocytes. Functionally, however, overexpression of miR-181c leads to an increase in both mitochondrial respiration and reactive oxygen species generation likely because of increased mt-COX2 mRNA and protein content, perhaps by compensating for reduced mt-COX1. Nevertheless, this study reveals the potential important role of miRNAs in the regulation of mitochondrial gene expression and function (Das et al., 2012). Finally, miR-181c expression was increased in human cardiac samples from individuals with ventricular septal defects relative to healthy subject controls. Overexpression of miR-181c reduced the expression and 3′-UTR activity of bone morphogenetic protein receptor type II, a receptor for bone morphogenetic protein ligands implicated in septal formation in the heart. These findings raise the possibility that cardiac miR-181c may be associated with the pathogenesis of ventricular septal defects (Li et al., 2013).

Osteopontin (OPN) is a multifunctional protein highly expressed in bone and various cell types, including vascular smooth muscle cells (VSMCs), and involved in the development of atherosclerosis (Scatena et al., 2007). For example, Ang II-induced atherosclerosis is attenuated in OPN-deficient mice (Bruemmer et al., 2003). In VSMCs, Ang II decreased miR-181a expression, and overexpression of miR-181a inhibited Ang II-induced OPN expression and the adhesion of VSMCs to collagen, suggesting miR-181a might participate in the pathophysiology of atherosclerosis through modulation of OPN-mediated function of VSMCs (Remus et al., 2013).

Role of miR-181 family members in leukocytes

The recruitment and accumulation of leukocytes in the vessel wall is a hallmark in the initiation and progression of acute and chronic inflammatory disease states. MiR-181 family members have been shown to play key roles in leukocyte cell differentiation and function. We summarize below the role of miR-181 in various leukocyte subsets and provide implications for vascular inflammation.

MiR-181a and MiR-181b in leukocytes

One of the first reported functions for miR-181a in leukocytes was its regulatory effect in B cell differentiation. MiR-181a was preferentially expressed in the B-lymphoid cells of mouse bone marrow, and its expression increased during B cell lineage commitment (Chen et al., 2004). Ectopic expression of miR-181a in hematopoietic progenitor cells altered lineage differentiation resulting in a substantial increase in the proportion of B-lineage cells (CD19+) in vitro and in vivo (Chen et al., 2004). MiR-181b also affects somatic hypermutation and class switch recombination in B cells, processes that are initiated by activation-induced cytidine deaminase (AID) and generate unique B-cell antibodies (de Yebenes et al., 2008). MiR-181b inhibits class switch recombination by binding to the 3′-UTR of AID resulting in reduced AID mRNA and protein levels (de Yebenes et al., 2008). MiR-181a also plays a distinct role in T cell maturation. Distinct miRNAs are known to be expressed at different stages of T cell development. In particular, miR-181a was induced at the CD4(+)CD8(+) double-positive stage and inhibited the expression of B-cell lymphoma 2, CD69, and T-cell receptor (TCR) which are all involved in positive selection and T cell maturation (Neilson et al., 2007). MiR-181a also functions as an endogenous regulator of T cell sensitivity and selection. Increasing expression of miR-181a in mature T cells increased T-cell sensitivity; in contrast, decreasing expression of miR-181a reduced T-cell sensitivity and impaired both positive and negative selection(Li et al., 2007). MiR-181a regulates T cell sensitivity, in part, by decreasing expression of multiple phosphatases and a reduction of TCR signaling threshold (Li et al., 2007). MiR-181a also plays a role in regulating positive section in the thymus. Inhibition of miR-181a produced overly reactive T cells suggesting that miR-181a functions to eliminate high affinity T cells during positive selection (Ebert et al., 2009). MiR-181a may also affect senescence of T cell sensitivity (Li et al., 2012). In CD4+ T cells, age-dependent defects in the extracellular signal-related kinase (ERK) pathway were regulated by increased levels of dual specific phosphatase 6 (DUSP6). Consequently, DUSP6 levels were shown to increase with age due to attenuated inhibition by miR-181a. Restoration of miR-181a levels reduced DUSP6 expression in CD4+ T cells in elderly individuals. Furthermore, DUSP6 inhibition using miR-181a overexpression, siRNA ‘knockdown’ to DUSP6, or DUSP6 allosteric inhibitors increased activation markers, increased proliferation, and supported T helper type 1 differentiation. This suggests that miR-181a may be a useful target in increasing the effectiveness of vaccination in the elderly (Li et al., 2012). Thus, current evidence suggests miR-181a plays an important regulatory role in B-cell differentiation, positive and negative selection during T cell development, and T cell sensitivity—effects that have implications for fine-tuning the immune response.

Recently, the function of miR-181a1/b1 in T cell development was demonstrated in knockout mice. PI3K signaling is critical for anabolic metabolism to support high proliferation rate and elevated biosynthetic demands during T cell development. In natural killer T cells (NKT), miR-181a1/b1 was found to be a non-redundant determinant of PI3K signaling strength through controlling PTEN expression. MiR-181a1/b1-deficient mice exhibited increased PTEN expression, impaired PI3K signaling, including downstream mTOR and FoxO signaling, dysregulated cellular metabolism, and severe defects in lymphoid development with severe deficiency of mature NKT in the thymus and periphery (Henao-Mejia et al., 2013). Invariant NKT cells (iNKT) are a subset of NKT cells expressing a rearranged TCR with a semi-invariant TCRα chain and a restricted set of TCRβ chains. MiR-181a1/b1 deficiency led to a dramatic reduction of iNKT cells in thymus and in the periphery. In addition, deletion of miR-181a1/b1 caused increased expression of multiple negative regulators of TCR signaling, and consequently resulted in an altered TCR repertoire of iNKT cells, and reduced responsiveness toward extrinsic signals. These data demonstrated that miR-181a1/b1 controls iNKT cell differentiation through setting a threshold for agonist selection (Zietara et al., 2013).

MiR-181a and miR-181b were also shown to have an important regulatory role in monocytes especially in the context of inflammation in obesity (Hulsmans et al., 2012). Inflammatory TLR/NF-κB signaling in monocytes contributes to inflammation in obesity which is associated with increased risk of metabolic syndrome and coronary artery disease. Consistent with our previous report (Sun et al., 2012), miR-181a, miR-181b, and miR-181d were all shown to be possible regulators of TLR/NF-κB signaling via in silico target prediction analysis and their expression was significantly reduced in monocytes of obese patients, and normalized in monocytes after weight loss to levels achieved in lean subjects (Hulsmans et al., 2012). Furthermore, miR-181a was retrospectively associated with increased incidence of metabolic syndrome and coronary artery disease even after adjustment for traditional risk factors, obesity, and metabolic syndrome. Thus, reduced expression of miR-181 family members in obese subjects may serve as potential biomarkers of metabolic syndrome and coronary artery disease (Hulsmans et al., 2012). In peripheral blood mononuclear cells of type 2 diabetic patients taking resveratrol enriched grape extract for 12 months, reduced expression of pro-inflammatory cytokines including CCL3, IL-1β and TNF-α was associated with increased expression of miR-181b along with five other miRNAs (Tome-Carneiro et al., 2013). Thus, these correlative studies along with the known role that miR-181b exerts on dampening downstream NF-κB signaling (Sun et al., 2012) suggests that miR-181b plays a protective role in vascular inflammation and potentially cardiovascular disease. Future studies will be required to examine a potential causal relationship of miR-181 with insulin resistance, diabetes, and atherosclerosis.

MiR-181a may also modulate inflammation by regulating dendritic cell (DC) function. In atherosclerosis, oxidized LDL (ox-LDL) activates DCs, which facilitates the inflammatory response (Bobryshev, 2010). MiR-181 expression levels were increased in CD11c(+) DCs of ApoE-deficient mice with hyperlipidemia (Wu et al., 2012). In cultured bone marrow-derived DCs, ox-LDL increased DC maturation and induced miR-181a expression. Ox-LDL upregulated DC maturation markers (CD83 and CD40) and inflammatory cytokines (IL-6 and TNF-α), while downregulated the anti-inflammatory cytokine IL-10 in bone marrow-derived DCs. Overexpression of miR-181a reduced the ox-LDL-mediated increase in CD83 and CD40, reduced secretion of IL-6 and TNF-α, and increased IL-10 expression. These effects were mediated in part by miR-181a targeting the 3′- UTR of c-Fos. This evidence suggests that ox-LDL induced inflammation is repressed by miR-181a in a negative feedback fashion (Wu et al., 2012).

MiR-181c and MiR-181d in leukocytes

MiR-181c also has documented roles in the inflammatory response. For example, miR-181c may play a role in CD4+ T cell activation (Xue et al., 2011). In Jurkat T cells and PBMCs, miR-181c expression is reduced during CD4+ T cell activation. Furthermore, miR-181c acts as a suppressor of CD4+ T cell activation by binding to the 3′-UTR of IL-2 (Xue et al., 2011). In addition, miR-181c may also play a role in immunosenescence in the context of aging and chronic heart failure (CHF). Older individuals exhibited reduced expression of miR-181 family members and lowered lymphocyte counts including cytotoxic T cells and B cells in peripheral blood. In particular, miR-181c was further reduced in age-matched CHF patients (Seeger et al., 2013). Because miR-181c levels were particularly reduced in subjects with CHF, it will be of interest to determine in future prospective studies whether miR-181c may serve as biomarker for reduced immune function in CHF (Seeger et al., 2013).

The acute stress response, such as by infection or surgery, may trigger systemic or intrathymic production of glucocorticoids that regulates lymphocyte development and triggers an apoptotic program (Ashwell et al., 2000; Vacchio and Ashwell, 2000; Vacchio et al., 1994). The miR-181 family was among 18 other thymic miRNAs found to be dysregulated in response to LPS or the synthetic glucocorticoid dexamethasone in thymocytes (Belkaya et al., 2011). Strikingly, miR-181d expression was reduced up to 15-fold in response to LPS or dexamethasone. MiR-181d uniquely targeted the 3′-UTR of leukemia inhibitory factor and reduced its expression compared to other miR-181 family members. These observations implicate the miR-181 family especially miR-181d in thymopoiesis during acute stress response in the thymus (Belkaya et al., 2011).

Conclusion and Future direction

The studies discussed above implicate critical roles for miR-181 family members in cardiovascular inflammation and immune cell homeostasis. The emerging role for miR-181 family members in regulating cellular function of ECs and leukocytes in particular—two critical partners in vascular inflammation—indicate that miR-181 will likely have a central role in both acute and chronic inflammatory disease states such as atherosclerosis, type 2 diabetes, and obesity. These observations also underscore several important and unresolved issues. First, given the anti-inflammatory function of miR-181b in ECs (Sun et al., 2012), future studies will be required to identify upstream mechanisms governing the expression of miR-181b in endothelial cells in response to pathophysiological stimuli and pharmacological agents. Interrogation of the upstream regulatory ‘promoter’ regions of miR-181b and other family members may inform how to control or fine-tune miR-181 expression. Second, the basis for cell-type specific expression of individual miR-181 family members and, in some cases, their ability to target specific genes requires further investigation. Finally, future gain- and loss-of-function studies will certainly be required to directly study the function of miR-181 family members in chronic inflammatory disease states such as atherosclerosis, type 2 diabetes, and obesity—conditions in which vascular inflammation is a hallmark. In this regard, systemic delivery of miR-181 family members, especially miR-181b, may provide a novel therapeutic approach to treat these chronic inflammatory diseases in which miR-181 expression is reduced.