Taming endothelial activation with a microRNA

Inflammation plays an essential role in vascular pathologies, including those associated with sepsis and atherosclerosis. Identifying negative regulators of inflammatory signaling pathways may provide novel therapeutic targets for these diseases. In this issue of the JCI, Sun et al. show that in endothelial cells, microRNA-181b (miR-181b) plays a vital role in controlling inflammation by targeting importin-α3, a regulator of NF-κB nuclear import. These findings provide compelling evidence that modulation of microRNAs may be a useful therapeutic approach for inflammatory vascular diseases.

The vascular endothelium forms the interface between blood and tissues and plays a critical and active role in maintaining blood vessel and tissue homeostasis as well as a nonthrombotic and nonadhesive vascular surface. Endothelial cell activation by proinflammatory stimuli promotes leukocyte recruitment from the blood into extravascular tissues and thus contributes to the pathogenesis of various inflammatory diseases (1). Multiple signaling pathways participate in this process, but the NF-κB signaling pathway plays a particularly central role. NF-κB signal transduction in endothelium culminates in the expression of multiple proinflammatory genes, including cell adhesion molecules such as E- and P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1), as well as various chemokines and cytokines. Since endothelial cell activation contributes to the pathogenesis of multiple inflammatory and immune conditions, targeting NF-κB signal transduction is an attractive potential therapeutic strategy. However, enthusiasm should be tempered because a subset of NF-κB target genes promotes cell survival under conditions of stress. It is also important to target the “right” cells because inhibition of NF-κB signaling in endothelial cells reduces atherosclerosis, whereas inhibition in macrophages promotes cell death and exacerbates atherosclerosis (2, 3). In this issue of the JCI, Sun et al. (4) explore a novel microRNA-based approach to regulating NF-κB signaling and provide insights into its therapeutic utility.

The NF-κB/Rel family consists of protein homo- and heterodimers, each with different DNA binding and activation specificity (5). The p50/p65 heterodimer is the prototypic NF-κB and is predominant in endothelial cells. In quiescent cells, NF-κB is predominantly localized in the cytoplasm, where it is retained through association with an inhibitor protein (IκB) (ref. 5 and Figure 1). There are several IκBs, and they associate preferentially with different NF-κB dimers; IκBα associates primarily with the p50/p65 heterodimer. The binding of an IκB masks the classical nuclear localization sequence (NLS) of each NF-κB subunit and inhibits import to the nucleus.

Figure 1

MicroRNA-based regulation of NF-κB signaling and endothelial activation. Multiple inflammatory stimuli lead to activation of the IKK complex, which phosphorylates IκB and promotes its polyubiquitination and proteasomal degradation. NF-κB proteins (such as p50/p65 heterodimers) that are released from IκB are imported into the nucleus via their nuclear localization signals, where they activate the transcription of proinflammatory genes, including vascular adhesion molecules (i.e., Vcam1, Icam1, Sele). Nuclear import of NF-κB is facilitated by importin proteins, including importin-α3. MicroRNAs bind to the 3ι UTRs of target mRNAs and inhibit their stability and/or translation. miR-181b elicits an antiinflammatory effect in endothelial cells by repressing importin-α3 expression, thereby inhibiting nuclear import of NF-κB. In addition, an endothelial-specific microRNA, miR-126, inhibits the expression of VCAM-1, and inflammation induces the expression of several microRNAs, including miR-31, miR-17-3p, and miR-146a, which participate in negative feedback loops. miR-31 negatively regulates E-selectin, miR-17-3p represses ICAM-1 expression, and miR-146a targets adaptor molecules (i.e., IRAK1, TRAF6) that are involved in inflammatory signal transduction. miR-10a, which is reduced in regions of blood vessels that are exposed to disturbed flow, represses MAP3K7 (TAK1) and β-TRC, which promote IκB degradation.

Diverse stimuli, including ligand binding to proinflammatory cytokine receptors, TLR, and other receptors, such as RAGE, lead to activation of IκB kinases and NF-κB signaling via the canonical pathway (5). In this signaling cascade, IκB is phosphorylated on 2 conserved serines (S-32 and S-36 on IκBα), polyubiquitinated, and targeted for degradation by the 26S proteasome. This unmasks the NLS of NF-κB subunits and enables transport to the nucleus. Nuclear transport of proteins with a classical NLS is mediated by a heterodimeric complex made up of proteins from the importin-α and importin-β families, where the importin-α binds to the protein cargo and the importin-β mediates interaction with the nuclear pore complex (6). When the complex reaches the nucleus, it binds RanGTP, which induces a conformational change that dissociates it. Of the 6 human importin-α isoforms, only α3 and α4 are capable of binding the NLS of p50 and p65 and mediating nuclear import (7). In the nucleus, p50/p65 heterodimers recognize specific nucleotide sequences and transactivate gene expression through interactions with cofactors, other transcription factors, and histone acetyltransferases (5).

NF-κB signaling is autoregulated, since activation of this pathway induces the expression of a number of proteins that function as inhibitors of NF-κB activation. This includes rapid upregulation of IκBα expression and replenishment of its cytoplasmic pool. Unbound IκBα contains an exposed NLS and is imported into the nucleus, where it associates with DNA-bound NF-κB. Since IκBα also contains a nuclear export sequence, it binds exportins that transport the NF-κB/IκBα complex back to the cytoplasm (5). Thus, the NF-κB pathway is tightly regulated to control the intensity and duration of an inflammatory response.

MicroRNAs are small noncoding RNAs (~21 nucleotides in length) that bind primarily to the 3ι UTR of mRNAs and decrease their stability and/or inhibit translation (8). Recently, several microRNAs have been implicated in the regulation of the inflammatory response in endothelial cells (Figure 1). For example, miR-126, an endothelial-specific microRNA (9), inhibits the expression of VCAM-1 and antagonizes the binding of leukocytes to endothelial cells in vitro (10). Additionally, miR-31 and miR-17-3p are induced following exposure of endothelial cells to TNF-α, and these microRNAs negatively regulate E-selectin and ICAM-1 expression, respectively, thus acting in a negative feedback loop to restrain endothelial cell activation (11). miR-146a is another of a microRNA that participates in a negative feedback loop that tempers inflammatory signaling. In monocytes, miR-146a is transcriptionally induced via NF-κB binding to its promoter following activation of TLR signaling, and it represses upstream components of the TLR pathway, including IRAK1 and TRAF6 (12). The involvement of this microRNA in endothelial cell activation has not been addressed.

Disturbed blood flow promotes inflammation and atherosclerosis, in part by priming of canonical NF-κB signaling (13). Interestingly, miR-10a expression is lower in regions of the aorta that are exposed to disturbed flow (14). This microRNA regulates the expression of mitogen-activated kinase kinase kinase 7 (MAP3K7;TAK1) and β-transducin repeat-containing gene (β-TRC), both of which facilitate IκBα degradation. Inhibition of miR-10a therefore enhances NF-κB–dependent adhesion molecule expression in endothelial cells (14).

The findings of Sun et al. (4) provide an additional example of the intersection between microRNAs and the NF-κB pathway in endothelial cells. The authors elegantly demonstrate that miR-181b plays a key role in controlling endothelial activation. This is mediated by the regulation of importin-α3, a protein that facilitates the nuclear import of p50/p65. Using miR-181b mimetics in vivo, they show promising protective effects in a murine model of sepsis that are consistent with a previous study in which endothelial cells expressed a dominant negative IκBα (15). However, it remains to be seen whether over-expression of miR-181b can be sustained to provide protection against chronic inflammation. This is particularly relevant for chronic inflammatory vascular diseases such as atherosclerosis. Sun et al. additionally observed reduced levels of circulating miR-181b in patients with sepsis (4). Their data provide a rationale for targeting endothelial cell NF-κB signal transduction in septic patients. However, for this approach to be practical in patients, therapy would have to be effective when administered after the onset of sepsis. Whether miR-181b mimetics will be effective at later stages of sepsis is not addressed in the current body of work, and experiments in mouse and nonhuman primate models will be required before this treatment can be attempted in human clinical trials.

It is now apparent that several microRNA-based regulatory networks converge on NF-κB–dependent inflammatory pathways (refs. 4, 10, 11, 14, and Figure 1) and may contribute to diseases of the vasculature. Therapeutic manipulation of microRNA function is therefore being explored. Additionally, microRNAs can be detected in the circulation, and their expression is altered in disease conditions such as myocardial infarction (16) and septic shock (4). Therefore, microRNA expression may be utilized as a biomarker. Antisense microRNA inhibitors (anti-miRs) provide potent and long-term antagonism of microRNA function following intravenous or subcutaneous injection in animal models. For example, antagonism of miR-33a and miR-33b — which repress a network of genes involved in fatty acid oxidation and synthesis as well as the cholesterol transporter ABCA1 — enhances the level of HDL while suppressing VLDL levels in nonhuman primate models (17).

The first clinical trial using an anti-miR approach is currently underway, targeting miR-122 (18), which was previously shown to be involved in HCV replication (19).Santaris Pharma A/S is now performing phase II clinical trials utilizing miR-122 anti-miRs in HCV patients, and this approach appears to provide prolonged antiviral activity (18). Examples of therapeutic microRNA overexpression using microRNA mimetics are less common, but these approaches have shown some benefit in animal models (20). As opposed to anti-miRs, which can be administered globally to target cell-restricted microRNAs, such as miR-122 (liver) or miR-126 (endothelium), microRNA mimetics require cell- or tissue-specific delivery to reduce unintended side effects of microRNA overexpression in nontarget cells. This remains a major technical hurdle for these approaches.

Interestingly, Sun et al. (4) show that intravenous delivery of miR-181b mimetics results in overexpression of miR-181b in the intimal layer of blood vessels (i.e., endothelium), but not in the media and adventitia. Further experiments will be required to fully appreciate the cell and tissue distribution of injected miR-181b mimetics and the consequences of miR-181b overexpression in nonendothelial cells. It is intriguing that the effects of miR-181b treatment appear to be restricted to endothelial cells, since no effect on NF-κB–dependent gene expression was observed in peripheral blood mononuclear cells from miR-181b–injected mice. The molecular mechanisms for this endothelial specificity are not clear at present, since importin-α3 is presumably expressed in many cell types.

Targeting miR-181b to control endothelial activation appears to be a very promising therapeutic approach, and subsequent work will define whether this microRNA is able to effectively tame inflammation in the setting of human vascular disease and/or sepsis.

Research in the laboratories of J.E. Fish and M.I. Cybulsky is supported by operating grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research.

Address correspondence to: Jason E. Fish or Myron I. Cybulsky, Toronto General Research Institute, University Health Network, MaRS Centre, Toronto Medical Discovery Tower, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.581.7495; Fax: 416.581.7484; E-mail: jason.fish@utoronto.ca (J.E. Fish). Phone: 416.581.7483; Fax: 416.581.7484; E-mail: myron.cybulsky@utoronto.ca (M.I. Cybulsky).

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(6):1967–1970. doi:10.1172/JCI63818

See the related article at MicroRNA-181b regulates NF-κB–mediated vascular inflammation.

1. Pober JS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev Immunol. 2007;7(10):803–815.
   View this article via: PubMed Google Scholar
2. Gareus R, et al. Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis. Cell Metab. 2008;8(5):372–383.
   View this article via: PubMed CrossRef Google Scholar
3. Kanters E, et al. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. J Clin Invest. 2003;112(8):1176–1185.
   View this article via: JCI PubMed Google Scholar
4. Sun X, et al. MicroRNA-181b regulates NF-κB–mediated vascular inflammation. J Clin Invest. 2012;122(6):1973–1990.
   View this article via: JCI Google Scholar
5. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 2007;8(1):49–62.
   View this article via: PubMed CrossRef Google Scholar
6. Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH. Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem. 2007;282(8):5101–5105.
   View this article via: PubMed CrossRef Google Scholar
7. Fagerlund R, Kinnunen L, Kohler M, Julkunen I, Melen K. NF-{kappa}B is transported into the nucleus by importin {alpha}3 and importin {alpha}4. J Biol Chem. 2005;280(16):15942–15951.
   View this article via: PubMed CrossRef Google Scholar
8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–233.
   View this article via: PubMed CrossRef Google Scholar
9. Fish JE, et al. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell. 2008;15(2):272–284.
   View this article via: PubMed CrossRef Google Scholar
10. Harris TA, Yamakuchi M, Ferlito M, Mendell JT, Lowenstein CJ. MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1. Proc Natl Acad Sci U S A. 2008;105(5):1516–1521.
    View this article via: PubMed CrossRef Google Scholar
11. Suarez Y, Wang C, Manes TD, Pober JS. Cutting edge: TNF-induced microRNAs regulate TNF-induced expression of E-selectin and intercellular adhesion molecule-1 on human endothelial cells: feedback control of inflammation. J Immunol. 2010;184(1):21–25.
    View this article via: PubMed CrossRef Google Scholar
12. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A. 2006;103(33):12481–12486.
    View this article via: PubMed CrossRef Google Scholar
13. Hajra L, Evans AI, Chen M, Hyduk SJ, Collins T, Cybulsky MI. The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation. Proc Natl Acad Sci U S A. 2000;97(16):9052–9057.
    View this article via: PubMed Google Scholar
14. Fang Y, Shi C, Manduchi E, Civelek M, Davies PF. MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro. Proc Natl Acad Sci U S A. 2010;107(30):13450–13455.
    View this article via: PubMed Google Scholar
15. Ye X, Ding J, Zhou X, Chen G, Liu SF. Divergent roles of endothelial NF-kappaB in multiple organ injury and bacterial clearance in mouse models of sepsis. J Exp Med. 2008;205(6):1303–1315.
    View this article via: PubMed CrossRef Google Scholar
16. Wang GK, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 2010;31(6):659–666.
    View this article via: PubMed Google Scholar
17. Rayner KJ, et al. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature. 2011;478(7369):404–407.
    View this article via: PubMed CrossRef Google Scholar
18. van Rooij E, Purcell AL, Levin AA. Developing microRNA therapeutics. Circ Res. 2012;110(3):496–507.
    View this article via: PubMed CrossRef Google Scholar
19. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005;309(5740):1577–1581.
    View this article via: PubMed Google Scholar
20. Trang P, et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther. 2011;19(6):1116–1122.
    View this article via: PubMed Google Scholar