MicroRNA-181b regulates NF-κB–mediated vascular inflammation (original) (raw)
miR-181b expression in ECs is regulated by TNF-α. In an attempt to identify how proinflammatory stimuli regulate endothelial function, microarray miRNA profiling studies were undertaken using RNA from HUVECs exposed to vehicle alone or TNF-α for 4 hours, and reduced expression of miR-181b was noted (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI61495DS1). Using real-time PCR analysis, we verified that miR-181b was rapidly reduced in response to TNF-α at 1 and 4 hours by 31% and 24%, respectively (Figure 1A), whereas at 24 hours, miR-181b expression increased before returning to baseline (Supplemental Figure 1B). miR-181b belongs to the miR-181 family, which comprises 4 mature miRNAs: miR-181a, miR-181b, miR-181c, and miR-181d. These mature sequences are encoded by 6 primary miRNA sequences located on 3 different chromosomes. The expression of miR-181b was determined to be about 12-fold higher than that of miR-181a and 274-fold higher than that of miR-181c by real-time PCR (Figure 1B). Since the level of primary miR-181d was very low (data not shown), we did not examine the level of this mature miR-181d. Collectively, these data suggest that miR-181b is the dominantly expressed miR-181 family member in HUVECs and that its expression is rapidly reduced in response to stimulation by the inflammatory cytokine TNF-α.
miR-181b suppresses TNF-α–induced proinflammatory gene expression in HUVECs. (A) Real-time qPCR analysis of miR-181b in response to TNF-α (10 ng/ml) in HUVECs. (B) Real-time qPCR analysis of miR-181a, miR-181b, and miR-181c in HUVECs. Numbers over bars indicate fold change relative to miR-181c. (C) Western blot analysis of VCAM-1, E-selectin, and ICAM-1 in HUVECs transfected with miRNA negative control (NS-m) or miR-181b mimics (181b-m), miRNA inhibitor negative control (NS-i), or miR-181b inhibitor (181b-i), respectively, after treatment with 10 ng/ml TNF-α for 8 hours. Densitometry was performed and fold change of protein expression is shown below the corresponding band. (D) Real-time qPCR analysis of VCAM-1, E-selectin, and ICAM-1 mRNA levels in HUVECs transfected with miRNA negative control, miR-181b mimics, miRNA negative control, or miR-181b inhibitor, and treated with 10 ng/ml TNF-α for the indicated times. (E) ELISA analysis of elaborated VCAM-1, E-selectin, and ICAM-1 protein levels in cell culture medium 16 hours after TNF-α (10 ng/ml) treatment. HUVECs were transfected as indicated in A. (F) miR-181b regulates the adhesion of THP-1 cells to TNF-α–activated HUVECs. Photo images of THP-1 cells adhering to HUVECs transfected with miRNA negative control or miR-181b mimics, miRNA inhibitor negative control, or miR-181b inhibitor with or without 10 ng/ml TNF-α treatment for 4 hours. #P < 0.05; *P < 0.01. Scale bars: 100 μm. All values represent mean ± SD.
miR-181b inhibits TNF-α–induced expression of adhesion molecules and inhibits leukocyte adhesion to activated EC monolayers. To assess the potential role of miR-181b in endothelial activation, we examined the effect of miR-181b on TNF-α–induced gene expression in HUVECs by using gain- and loss-of-function experiments. Overexpression of miR-181b inhibited TNF-α–induced VCAM-1, E-selectin, and ICAM-1 protein expression by 78%, 54%, and 44%, respectively, while miR-181b inhibitors (complementary antagonist) increased their expression by 122%, 81%, and 48%, respectively (Figure 1C and Supplemental Figure 1G). In agreement with these results, the mRNA levels of VCAM-1, E-selectin, and ICAM-1 were lower in cells overexpressing miR-181b than in cells overexpressing the miRNA negative control; moreover, these mRNA levels were higher in the presence of the miR-181b inhibitor (Figure 1D). After 1 hour of TNF-α treatment, cells overexpressing miR-181b exhibited reduced mRNA levels of VCAM-1, E-selectin, and ICAM-1 (by 69%, 65%, and 57%, respectively); after 4 hours of TNF-α treatment, the levels were 74%, 41%, and 17%, respectively. In contrast, in cells transfected with miR-181b inhibitors, TNF-α–induced VCAM-1 mRNA was increased by 62% after 1 hour of TNF-α treatment, and after 4 hours of TNF-α treatment, VCAM-1, E-selectin, and ICAM-1 mRNA levels were increased by 35%, 52%, and 34%, respectively (Figure 1D). The effects of miR-181b on levels of soluble VCAM-1, E-selectin, and ICAM-1 in the culture medium, as measured by ELISA, were also consistent with its effects on the mRNA and protein expression of these adhesion molecules (Figure 1E). Likewise, miR-181b also reduced VCAM-1 expression at both the protein and mRNA levels in HUVECs in response to LPS treatment (Supplemental Figure 1, C–E). Similar effects on VCAM-1 expression were observed for miR-181a (data not shown). Considering our finding that VCAM-1 expression was most sensitive to miR-181b, we determined whether the VCAM-1 gene might be a direct target of the miRNA. However, overexpression of miR-181b did not alter the luciferase activity of a VCAM-1 3′ UTR construct, suggesting that the VCAM-1 3′ UTR is not directly targeted by miR-181b (Supplemental Figure 1F). Since VCAM-1, E-selectin, and ICAM-1 are typical proinflammatory molecules induced by TNF-α (14, 15, 36–38), these data suggested that miR-181b may be involved in the regulation of EC activation. In response to EC activation, adhesion molecules, such as VCAM-1, E-selectin, and ICAM-1, act to initiate, promote, and sustain leukocyte attachment to the vascular endothelium. To determine the functional consequence of miR-181b effects on adhesion molecule expression, we employed an in vitro cell adhesion assay to assess leukocyte-EC interactions. As expected, TNF-α treatment markedly increased the adhesion capabilities of THP-1 cells to HUVECs transfected with miRNA negative control. However, adhesion was markedly reduced (by 44%) with miR-181b overexpression, whereas inhibition of miR-181b increased the adherence by 50% (Figure 1F). Taken together, these findings suggest that miR-181b is able to negatively affect the expression of key adhesion molecules induced by proinflammatory stimuli and that miR-181b dynamically regulates leukocyte adhesion to stimulated EC monolayers.
miR-181b suppresses TNF-α–induced expression of adhesion molecules in vivo. We next investigated whether systemic administration of miR-181b could inhibit TNF-α–induced gene expression in vivo. To assess the effects of miR-181b mimics on TNF-α–induced expression of VCAM-1 in vivo, miR-181b or a miRNA negative control (1 nmol/mouse) was admixed with atelocollagen and tail vein injected 24 hours prior to TNF-α treatment. VCAM-1 protein expression was first examined in lung tissues. At 4 hours after TNF-α i.p. injection, VCAM-1 was found to be induced by approximately 3.4-fold in lung tissues in the presence of NS control mimics. In contrast, administration of miR-181b potently reduced the induction of VCAM-1 protein expression (Figure 2A) and VCAM-1 mRNA expression in lung, aorta, heart, liver, and spleen (Figure 2B). The expression of E-selectin mRNA was also significantly reduced in lung, liver, and spleen (Supplemental Figure 2A), whereas ICAM-1 mRNA was reduced only in spleen (Supplemental Figure 2B). To further verify the observed effects on VCAM-1 expression, sections of lung and descending aorta were examined by immunohistochemical techniques. The endothelium of lung and aorta from mice injected with miRNA negative control displayed robust VCAM-1 expression in response to TNF-α (Figure 2, C–E). In contrast, systemic administration of miR-181b mimics reduced the induction of VCAM-1 expression in the endothelium in lung and aorta (by ∼86% and ∼80%, respectively) (Figure 2, C–E). Notably, the expression of miR-181b in the intima of aortae excised from mice injected with miR-181b was approximately 8-fold higher than in mice injected with miRNA negative control, as measured by real-time quantitative PCR (qPCR) (Supplemental Figure 2C). There were no significant differences in miR-181b expression levels in the media and adventitia excised from mice injected with miR-181b or miRNA negative control (Supplemental Figure 2C). Molecular ultrasound imaging using targeted microbubbles as a contrast agent has become a useful approach for noninvasive monitoring of the expression of adhesion molecules in vivo (39–42). We used this technique to further determine the effect of miR-181b on TNF-α–induced VCAM-1 expression in vivo. The innominate artery was selected for imaging as shown in Figure 2F. We first verified that VCAM-1–targeted MicroMarker ultrasound contrast showed a low level of signal-targeted enhancement in the innominate artery at baseline (Figure 2G). In response to TNF-α, compared with mice injected with control miRNA (Figure 2H), mice injected with miR-181b (Figure 2I) exhibited a marked reduction in differential signal–targeted enhancement values for VCAM-1 as quantified in Figure 2J. In summary, these data demonstrate that systemically administered miR-181b mimics were efficiently enriched in ECs and inhibited expression of TNF-α–induced adhesion molecules in vivo.
miR-181b represses TNF-α–induced proinflammatory gene expression in vivo. (A) Mice were i.v. injected with vehicle, miRNA negative control, or miR-181b mimics (50 μg/mouse). Twenty-four hours later, mice were treated with or without TNF-α for 4 hours, and lungs were harvested for Western blot analysis of VCAM-1 protein levels. Densitometry was performed and fold change of protein expression was quantified. (B) Experiments were carried out as described in A, and real-time qPCR analysis of VCAM-1 mRNA level in indicated tissues was performed. (C) VCAM-1 staining of lung and aorta sections. Mice were treated as in A. Scale bars: 25 μm (insets, 10 μm). (D and E) Quantification of VCAM-1 staining in lung and aortic endothelium, respectively. (A–E) Vehicle group (n = 3 mice), miRNA negative control group (n = 5 mice), miR-181b mimics group (n = 5 mice). Data represent mean ± SEM. (F) Ultrasound image shows region of interest (innominate artery) for in vivo VCAM-1 imaging using microbubble contrast. (G–I) Mice were injected with vehicle, miRNA negative control (n = 7), or miR-181b mimics (n = 6). Representative images show the differential targeted enhancement values for VCAM-1 expression detected by ultrasound before and after microbubble burst. (J) Quantification of differential targeted enhancement values for VCAM-1 expression in mice injected with miRNA negative control or miR-181b mimics. Data represent mean ± SEM. *P < 0.05.
miR-181b inhibits the NF-κB signaling pathway in activated ECs. In response to proinflammatory stimuli, both the NF-κB and MAPK pathways are involved in the inflammatory responses in ECs (21, 43). To determine whether miR-181b affects NF-κB activation, we first tested to determine whether miR-181b has any effect on the NF-κB concatemer and VCAM-1 promoter-reporter. As shown in Figure 3A, treatment of HUVECs with TNF-α induced the activity of both the NF-κB concatemer and the VCAM-1 promoter-reporter, and cotransfection of miR-181b significantly attenuated this induction. In contrast, inhibition of miR-181b potentiated TNF-α–induced activity (Figure 3A). We next explored the effect of miR-181b on NF-κB nuclear accumulation by immunostaining for p65. We observed a nearly 40% reduction in p65 nuclear staining in HUVECs transfected with miR-181b as compared with cells transfected with a miRNA-negative control (Figure 3B). After its release from the IκB complex, translocation of NF-κB from the cytoplasm to the nucleus is an essential step for the activation of NF-κB target genes (44, 45), an effect that can be revealed by detection of the p50 and p65 protein levels in cytoplasmic and nuclear fractions. As shown in Figure 3, C and D, HUVECs overexpressing miR-181b exhibited reduced p65 and p50 expression by 39% and 28%, respectively, in the nuclear fraction, whereas the cytoplasmic fraction had increased p65 and p50 expression by 35% and 37%, respectively. Importantly, we did not observe any significant differences between miR-181b and the miRNA negative control on the expression of upstream components of the NF-κB pathway, including phosphorylated IκBα (in the presence or absence of proteosome inhibitor MG-132), total IκBα, or phosphorylated IKKβ (Figure 3E and Supplemental Figure 3A) in response to TNF-α, suggesting that it is unlikely that miR-181b affects cell-surface receptors or activation of the IKK complex. Since several MAPKs have been implicated in TNF-α–induced expression of adhesion molecules (46–48), we next asked whether miR-181b had any effect on the activation of 3 MAPKs (ERK, p38, and JNK) in response to TNF-α. As shown in Supplemental Figure 3B, the phosphorylation of ERK, p38, and JNK was robustly induced and peaked at 15 minutes after TNF-α treatment. However, miR-181b overexpression had no inhibitory effect on their phosphorylation at 5, 15, and 30 minutes after TNF-α treatment. These data suggest that the inhibitory role of miR-181b on TNF-α–induced gene expression is primarily due to its effects on the NF-κB signaling pathway by repression of NF-κB nuclear translocation.
miR-181b inhibits the activation of the NF-κB signaling pathway. (A) Luciferase activity of reporters containing either the NF-κB concatemer or VCAM-1 promoter in HUVECs transfected with miRNA negative control or miR-181b mimics and miRNA inhibitor negative control or miR-181b inhibitor after 12 hours treatment with 10 ng/ml of TNF-α. #P < 0.05; *P < 0.01. Values represent mean ± SD; n = 3. (B) p65 staining in HUVECs transfected with miRNA negative control or miR-181b mimics. Thirty-six hours after transfection, cells were treated with 10 ng/ml TNF-α for 60 minutes and processed for immunostaining with antibodies against p65. Cells were stained with DAPI to visualize the nucleus. Cy3-conjugated miRNA negative control was transfected at 3 nM concentration to show transfection efficiency in both groups. Images were acquired by confocal microscopy from 3 independent experiments, and values represent mean ± SD. *P < 0.01. Scale bars: 20 μm. (C) The indicated proteins were detected in cytoplasmic or nuclear fractions prepared from HUVECs transfected with miRNA negative control or miR-181b mimics and treated with 10 ng/ml TNF-α for 1 hour. Densitometry was performed and fold change of p65 and p50 protein expression after normalization is shown below the corresponding band. Quantifications from 3 independent experiments were shown in D, and values represent mean ± SD. *P < 0.05. (E) Western blot analysis of phosphorylated IκBα or IKKβ in HUVECs transfected with miRNA negative control, or miR-181b mimics and treated with 10 μM MG-132 or DMSO for 2 hours followed by 10 ng/ml TNF-α for the indicated times.
miR-181b directly targets expression of importin-α3, a protein critical for NF-κB nuclear translocation. Previous studies have shown that NF-κBs are transported into the nucleus via a subset of importin-α molecules (49, 50). Because miR-181b overexpression had no effect on upstream phosphorylated IκBα or IKKβ expression, we hypothesized that miR-181b may inhibit downstream NF-κB signaling by targeting importin-α family members. There are 6 importin-α paralogs in humans (importin-α1, -α3, -α4, -α5, -α6, and -α7) that are characterized by distinct affinities to their substrates (51). Among these molecules, only importin-α3 and importin-α5 are predicted to be miR-181b targets, according to the algorithms of TargetScan (52), PITA (53), and miRanda (54). In miR-181b–overexpressing cells, importin-α3 expression, but not that of importin-α1 or importin-α5, was reduced by 40% and 25%, respectively, in the presence or absence of TNF-α (Figure 4A). Overexpression of miR-181b inhibited the activity of a luciferase reporter construct containing importin-α3 3′ UTR in a dose-dependent manner (Figure 4B). In contrast, the activity of luciferase constructs containing the 3′ UTR of importin-α1, -α4, or -α5 was not inhibited by overexpressed miR-181b (Figure 4C).
miR-181b reduces importin-α3 expression. (A) Western blot analysis of importin-α1, importin-α3, and importin-α5 in cells transfected with miRNA negative control or miR-181b mimics in the absence or presence of 10 ng/ml TNF-α. Mean ± SD, n = 3. *P < 0.05. (B) Normalized luciferase activity of a reporter containing the 3′ UTR of importin-α3 in cells cotransfected with increasing amounts of pcDNA3.1 empty vector or pcDNA3.1-miR-181b. *P < 0.01. (C) Normalized luciferase activity of a reporter containing the 3′ UTR of importin-α1, importin-α3, importin-α4, and importin-α5 cotransfected with either pcDNA3.1 empty vector or pcDNA3.1-miR-181b. *P < 0.01. (D) Normalized luciferase activity of a reporter containing 3′ UTR of importin-α3, predicted miR-181b–binding sites, or mutated miR-181b–binding sites. The reporter was cotransfected with either pcDNA3.1 empty vector or pcDNA3.1–miR-181b. *P < 0.05. (E) miRNP-IP analysis of enrichment of importin-α3 mRNA in HUVECs transfected with miRNA negative control or miR-181b mimics. *P < 0.01. (F) Luciferase activity of reporters containing the NF-κB concatemer in cells transfected with miRNA negative control or miR-181b mimics in the absence or presence of importin-α3 gene lacking its 3′ UTR. *P < 0.05. (G) Mice were i.v. injected with vehicle (n = 3 mice), miRNA negative control (n = 4 mice), or miR-181b mimics (n = 5 mice) and treated with TNF-α for 4 hours; aortas were harvested for importin-α3 staining. *P < 0.05. Scale bars: 20 μm (insets, 10 μm). (H) Western blot analysis of importin-α3 of lung ECs freshly isolated from mice treated with miRNA negative control or miR-181b mimics. Mean ± SD, n = 3. *P < 0.05. (I) Western blot analysis of importin-α3 of lung ECs freshly isolated from mice treated with miRNA inhibitor negative control or miR-181b inhibitor. Mean ± SD, n = 3. *P < 0.05.
The rna22 miRNA target detection and predication algorithm allows for seed mismatches between mature miRNA and targeting mRNA sequence (55) and has been successfully used to identify direct targets that were not predicted by other algorithms (56). To identify additional miR-181b–binding sites that may exist in the 3′ UTR of importin-α3, we applied the rna22 prediction algorithm and found 8 potential miR-181b–binding sites in the region of interest (Supplemental Figure 4A). Overexpression of miR-181b decreased luciferase activity by 31% and 20%, respectively, for luciferase-reporter constructs containing binding site 1 and site 2, but not for any of the other potential binding sites (Figure 4D). Site-directed mutations of binding site 1 and site 2 rescued the miR-181b–mediated inhibitory effects on both of these constructs (Figure 4D). While site 1 has imperfect complementarity to miR-181b, it is evolutionarily conserved across species (Supplemental Figure 4B). Interestingly, the mRNA level of importin-α3 was not altered by miR-181b overexpression (Supplemental Figure 4C), an effect indicating that the reduction of importin-α3 at the protein level is likely due to translation inhibition and not mRNA decay. To further verify that miR-181b directly targets importin-α3, we performed Argonaute2 (AGO2) micro-ribonucleoprotein IP (miRNP-IP) studies to assess whether importin-α3 mRNA is enriched in the RNA-induced silencing complex following miR-181b overexpression. An approximately 4-fold enrichment of importin-α3 mRNA was observed after AGO2 miRNP-IP in the presence of miR-181b, as compared with that with the miRNA-negative control (Figure 4E). In contrast, AGO2 miRNP-IP did not enrich the mRNA for Smad1, a gene that was not predicted to be an miR-181b target (Figure 4E). Moreover, expression of importin-α3 lacking its 3′ UTR was able to rescue the inhibitory effect of miR-181b on NF-κB activation (Figure 4F). Systemic delivery of miR-181b also reduced importin-α3 expression by 40% in aortic endothelium (Figure 4G) and nearly 50% in lung ECs (Figure 4H). Conversely, inhibition of miR-181b increased importin-α3 expression by approximately 40% in HUVECs (Supplemental Figure 4E) and approximately 50% in the lungs in vivo (Figure 4I). To explore whether “knockdown” of importin-α3 expression could “phenocopy” the inhibitory effects of miR-181b on NF-κB, we systemically delivered importin-α3 siRNA by tail-vein injections. As shown in Supplemental Figure 5, A and B, importin-α3 siRNA reduced _importin-α_3 mRNA and protein expression in freshly isolated lung ECs. Importantly, importin-α3 siRNA reduced NF-κB targets VCAM-1 and E-selectin (Supplemental Figure 5, C and D) and NF-κB activity in the lungs in vivo (Supplemental Figure 5E) as well as VCAM-1 and E-selectin expression in HUVECs in vitro (Supplemental Figure 5, F and G). Moreover, systemic delivery of importin-α3 lacking its 3′ UTR was capable of rescuing the miR-181b–mediated inhibitory effect on VCAM-1 and E-selectin expression and NF-κB activity in the lungs of mice treated with LPS (Supplemental Figure 5, H and I). In HUVECs, overexpression of importin-α3 lacking its 3′ UTR also rescued the miR-181b–mediated reduced expression for VCAM-1 and E-selectin compared with control (Supplemental Figure 5, J and K). Collectively, these data indicate that miR-181b inhibits the NF-κB signaling pathway by directly targeting importin-α3 expression.
miR-181b inhibited an enriched set of NF-κB–regulated genes in ECs. To systemically identify targets and biological processes regulated by miR-181b, we comparatively analyzed the gene expression profiles of HUVECs transfected with miRNA negative control or miR-181b by using Agilent whole human genome microarrays. Transfected HUVECs were treated with TNF-α for 4 hours, and total RNA was isolated and processed for gene chip analysis. Out of the approximately 44,000 transcripts screened, we determined that 841 genes were downregulated and 928 genes were upregulated by at least 1.5-fold in miR-181b–overexpressing cells as compared with control cells. Over 200 of those genes are known to be NF-κB regulated. Moreover, of 24 selected genes known to be associated with inflammatory disease states, all were inhibited by overexpression of miR-181b. These reduced gene expression changes were verified by qPCR analysis (Figure 5, A and B). Because the dominant-negative IκBα is a specific inhibitor of the canonical NF-κB signaling, we examined the gene expression profile of the 24 selected genes by real-time qPCR in HUVECs transduced in the presence of an adenovirus expressing the dominant-negative IκBα. We found that the expression of each of the 24 genes was reduced by expression of the dominant-negative IκBα in response to TNF-α treatment (Figure 5C), revealing a similar profile to cells overexpressing miR-181b mimics (Figure 5B). PAI-1, COX-2, CX3CL-1, and VCAM-1 were chosen to verify concordant directional changes at the protein level in miR-181b–overexpressing ECs (Figure 5D). To identify highly regulated biological processes in miR-181b–overexpressing cells, we performed gene set enrichment analysis (GSEA), a computational method that determines whether a defined set of genes shows significant differences between 2 biological states (57). We found 6 enriched biological processes that were significantly represented by the reduced genes in miR-181b–overexpressing cells: response to cytokine stimulus; positive regulation of cell migration; regulation of inflammatory response; inflammatory response; chemotaxis; and IKK/NF-κB cascade (Table 1). An abundance of targets that interconnected with the NF-κB signaling pathway was also observed using the Ingenuity web-based pathway analysis program (Supplemental Figure 6). Taken together, these data suggest that miR-181b selectively suppresses an enriched set of NF-κB–regulated genes and components of inflammatory signaling pathways in response to TNF-α in ECs.
Gene expression profiling in HUVECs transfected with miR-181b and bioinformatics analysis. (A) Relative gene expression of 24 TNF-α–regulated genes in HUVECs transfected with miRNA negative control or miR-181b mimics, as identified by microarray gene chip assay. Expression is presented as fold change relative to HUVECs transfected with miRNA negative control. Data shown are mean ± SD, n = 4. #, nonsignificant comparison; all other genes examined were significantly reduced by miR-181b overexpression (P < 0.05). (B) Real-time qPCR analysis of the genes listed in A. All genes examined were significantly reduced by overexpression of miR-181b (P < 0.05). (C) HUVECs infected with control virus or Ad-DN-IκBα were treated with TNF-α and harvested for real-time qPCR analysis of the genes listed in A. All genes examined were significantly reduced by Ad-DN-IκBα (P < 0.05). (D) Western blot analysis of CX3CL-1, PAI-1 (gene symbol, SERPINE1), COX-2 (gene symbol, PTGS2), and VCAM-1 in HUVECs transfected with miRNA negative control or miR-181b mimics. Values represent mean ± SD, n = 3. *P < 0.05.
Enriched GO biological processes downregulated in miR-181b overexpression cells, identified by GSEA at 25% FDR
miR-181b reduces LPS-induced EC activation, leukocyte accumulation, lung inflammation/injury, and mortality. Sepsis is a medical condition with high morbidity and mortality and increasing incidence over the past few decades (58). The endotoxin LPS is a component of the outer membrane of Gram-negative bacteria that plays a significant role in the pathogenesis of about 25% to 30% of sepsis cases (59). LPS activates the TLR-mediated signaling pathway, induces the release of critical proinflammatory cytokines including TNF-α, and elicits a systemic inflammatory response syndrome (SIRS) (60, 61). During sepsis, activation of the vascular endothelium plays a critical role in the recruitment of neutrophils and monocytes/macrophages as well as subsequent exacerbation of the inflammatory response (1, 62). To determine whether miR-181b contributes to this process, we assessed whether the level of endothelial miR-181b could be affected in response to LPS and whether in vivo overexpression of miR-181b could reduce leukocyte recruitment and EC activation in a systemic LPS mouse model of vascular inflammation. As shown in Supplemental Figure 7A, the expression of miR-181b was reduced in freshly isolated aortic intima by 50% and 47% after 4 hours of systemic treatment with LPS or TNF-α, respectively. In contrast, VCAM-1 mRNA was induced about 5.0-fold and 7.2-fold by TNF-α and LPS, respectively, in freshly isolated aortic intima (Supplemental Figure 7B). In vivo i.v. administration of miR-181b mimics reduced the induction of VCAM-1 expression in response to LPS by 60% (Figure 6, A and E). Analysis of lung sections taken from mice injected with miR-181b revealed a significant reduction of CD45-positive (common leukocyte antigen) and Gr-1–positive (predominantly neutrophil) leukocytes (by 54% and 58%, respectively) in response to LPS treatment (Figure 6, A, C, and D). Importantly, the number of Gr-1–positive leukocytes adherent to the pulmonary vascular endothelium was reduced 47% by miR-181b overexpression (Figure 6, G and H). We also observed reduced interstitial edema and lung injury scores in the lungs of mice in which miR-181b was overexpressed (Figure 6, A and B). Myeloperoxidase activity, reflecting the presence of peroxidase enzyme expressed most abundantly in neutrophils, was also reduced by miR-181b overexpression (Figure 6F). Finally, systemic delivery of miR-181b mimics in mice (2 injections before and 1 injection after the initiation of LPS-induced endotoxic shock) reduced mortality from endotoxemia by 75% over 4 days (Figure 6I). Taken together, these results demonstrate a critical role for miR-181b in endotoxin-induced endothelial activation and leukocyte accumulation and suggest that overexpression of miR-181b could represent a novel class of therapeutic avenues for limiting endotoxin-induced vascular inflammation and may ultimately have a beneficial effect in critical illness.
miR-181b reduces EC activation and leukocyte accumulation in LPS-induced lung inflammation/injury. (A) Mice were i.v. injected with vehicle, miRNA negative control, or miR-181b mimics and treated with or without LPS (40 mg/kg, i.p., serotype 026:B6) for 4 hours; lungs were harvested and stained for H&E, Gr-1, CD45, or VCAM-1 staining. Scale bars: 50 μm (insets, 20 μm). (B) Evaluation of lung injury 4 hours after LPS was determined by lung injury scoring. Each data point represents score from 1 section. n = 4 mice per group, and 3 sections per mouse were scored. *P < 0.05. (C) Quantification of CD45-positive cells. *P < 0.05. (D) Quantification of Gr-1 positive cells. *P < 0.05. (E) Quantification of VCAM-1 expression. *P < 0.05. n = 4 mice per group; values represent mean ± SD (C–E). (F) Mice were treated as in A. Lungs were harvested and assessed for MPO activity, and the value of the vehicle group was set to 1. Values represent mean ± SD, n = 6 mice per group. (G and H) Mice were treated as in A, and lungs were harvested for Gr-1 staining. Scale bars: 20 μm. Quantification shows the number of Gr-1–positive cells per mm vessel length. Values represent mean ± SD, n = 4. *P < 0.05. (I) Kaplan-Meier survival curves of: LPS-treated C57BL/6 mice (50 mg per kg, i.p., n = 10 to 11 per group) that were injected i.v. with vehicle (black circles), miRNA negative control (red squares), or miR-181b mimics (blue triangles) 48 hours before, 24 hours before, and 1.5 hours after LPS administration. *P < 0.05, 1-tailed log-rank test.
miR-181b inhibits the activation of the NF-κB pathway in vascular endothelium in vivo. In response to inflammatory stimuli, activation of the NF-κB pathway, which involves translocation of NF-κB heterodimers from cytoplasm to nucleus, is critical for vascular inflammation in vivo (24, 26). To determine whether miR-181b regulates NF-κB translocation in vivo, miR-181b was systemically administered by tail-vein injection, and mice were challenged with or without LPS. In mice not exposed to LPS, the majority of NF-κB p65 remained cytoplasmic in pulmonary ECs (Figure 7A). In contrast, NF-κB p65 colocalized more with DAPI-stained endothelial nuclei by immunofluorescence in mice challenged with LPS by i.p. injection (Figure 7A). Remarkably, the accumulation of NF-κB p65 in endothelial nuclei in response to LPS was reduced by 45% in the presence of systemically delivered miR-181b compared with mice injected with NS control miRNA (Figure 7A). Using a complementary approach, we also analyzed the effect of miR-181b on NF-κB activation in vivo in NF-κB transgenic mice, which express a luciferase reporter driven by an NF-κB–responsive promoter (63, 64). Indeed, systemic delivery of miR-181b reduced LPS-induced luciferase activity in lungs by 57% (Figure 7B), NF-κB target genes VCAM-1 and E-selectin in lungs by 43% and 59%, respectively (Figure 7, C and D), and VCAM-1 expression in isolated mouse lung ECs by 52% (Figure 7E). These effects were associated with approximately 4.5-fold overexpression of miR-181b in the lungs by real-time qPCR compared with mice injected with NS control miRNA (Figure 7F). Importantly, miR-181b reduced LPS-induced luciferase to a degree similar to that of adenoviral vectors expressing a dominant-negative IκBα, and no additional inhibitory effect of miR-181b was observed when delivered in combination with adenoviral vectors expressing a dominant-negative IκBα (Figure 7G). Finally, miR-181b overexpression had no effect on the expression of a panel of miRNAs in mouse lungs, which have been shown to regulate inflammatory genes or NF-κB activation, including miR-10a, miR-146a, miR-155, miR-31, and miR-17-3p (Supplemental Figure 8). These data suggest that systemic delivery of miR-181b inhibits endothelial NF-κB p65 nuclear translocation and, subsequently, the activation of NF-κB signaling pathway in vascular ECs in vivo.
Systemic delivery of miR-181b inhibits NF-κB activation in vivo. (A) Mice (n = 3–5 per group) were injected with vehicle, miRNA negative control, or miR-181b 3 times, and lungs were harvested on day 4 after 2 hours of 10 mg/kg LPS (026:B6, i.p.) for immunostaining with anti-p65 (red), anti-CD31 (green), and DAPI (blue). Arrows indicate differential p65 accumulation in nuclei of ECs after treatment. Scale bars: 20 μm (insets, 10 μm). Nuclear p65 was quantified in vascular ECs reflecting vehicle (n = 22 ECs), miRNA negative control (n = 40 ECs), and miR-181b mimics (n = 39 ECs). Mean ± SEM. *P < 0.01. (B) NF-κB luciferase transgenic mice (n = 4 per group) were injected with vehicle, miRNA negative control, or miR-181b as described in A. On day 4, 4 hours after 10 mg/kg LPS (026:B6), lungs were harvested for luciferase activity assay. Mean ± SD. *P < 0.05. (C and D) Real-time qPCR analysis of VCAM-1 or E-selectin. Mean ± SEM. *P < 0.05. (E) Western blot analysis of VCAM-1 in freshly isolated lung ECs from treated mice. Mean ± SEM, n = 2. *P < 0.05. (F) Real-time qPCR analysis of miR-181b expression in lungs after 4 hours LPS. Mean ± SD. *P < 0.01. (G) NF-κB luciferase transgenic mice (n = 6–8 per group) were injected with recombinant adenovirus Ad-GFP or Ad–DN-IκBα, followed by i.v. injection of vehicle, miRNA negative control, or miR-181b as described in A. Lungs were harvested on day 5 after 4 hours of 10 mg/kg LPS (026:B6) for luciferase activity assay and Western blot analysis. Mean ± SD. * P < 0.05.
Inhibition of miR-181b potentiated LPS-induced NF-κB–regulated gene expression and NF-κB activity in vivo. To determine whether the endogenous expression of miR-181b has any effect on NF-κB signaling and EC activation in vivo, mice were injected i.v. with miR-181b inhibitors in the presence of LPS. As shown in Figure 8, A and B, systemic delivery of miR-181b inhibitors reduced endogenous miR-181b expression by approximately 62% and potentiated LPS-induced VCAM-1 protein expression by approximately 1.9-fold in lung tissues. The expression of VCAM-1 and E-selectin mRNA also increased by approximately 1.9- and 1.5-fold, respectively (Figure 8, C and D). Furthermore, miR-181b inhibition enhanced NF-κB activity in lung lysates from NF-κB–luciferase transgenic mice treated with LPS (Figure 8E) and exacerbated lung injury, as shown by scoring (Figure 8G). Finally, in response to LPS, leukocyte accumulation in the lungs of mice treated with miR-181b inhibitors was markedly increased by immunohistochemistry studies compared with that in mice treated with NS control inhibitors (Figure 8, F, H, and I). These findings indicate that inhibition of endogenous miR-181b potentiates NF-κB signaling, EC activation, and lung inflammation in the presence of inflammatory stimuli, suggesting that reduced miR-181b expression may play a role in the pathogenesis of sepsis.
Inhibition of miR-181b potentiates LPS-induced proinflammatory gene expression in vivo. (A–E) NF-κB transgenic mice (n = 5 per condition) were i.v. injected with vehicle, miRNA inhibitor negative control, or miR-181b inhibitors (2 nmol/injection, 3 injections on consecutive days). Twenty-four hours after the last injection, mice were treated with or without LPS (10 mg/kg) for 4 hours, and lungs were harvested for different assays. (A) Real-time qPCR analysis of miR-181b expression. Mean ± SEM. (B) Western blot analysis of VCAM-1 protein expression. Densitometry was performed, and fold change of protein expression was quantified. Mean ± SEM. (C) Real-time qPCR analysis of VCAM-1 mRNA expression. Mean ± SEM. (D) Real-time qPCR analysis of E-selectin mRNA expression. Mean ± SEM. (E) Luciferase activity in lung lysates. Mean ± SD. (F–I) Mice (n = 3–5 per group) were treated as in A. (F) H&E, CD45, Gr-1 staining of lung sections. Scale bars: 50 μm. (G) Evaluation of lung injury 4 hours after LPS was determined by lung injury scoring. Each data point represents a score from 1 section. 2 or 3 sections per mouse were scored. (H) Quantification of CD45-positive cells. Mean ± SD. *P < 0.05. (I) Quantification of Gr-1–positive cells. Mean ± SD.
Circulating levels of miR-181b are reduced in patients with sepsis. Accumulating studies have reported that miRNA can be detected in the plasma and other body fluids (65–67). To examine the regulation of circulating miR-181b in an acute inflammatory disease, we determined the levels of miR-181b in plasma from patients with sepsis or with sepsis and acute respiratory distress syndrome (ARDS). As shown in Figure 9, A and B, the levels of circulating miR-181b were reduced by approximately 40% in patients with sepsis or sepsis plus sepsis/ARDS compared with control subjects admitted to the intensive care unit (ICU) without sepsis. In a multivariate logistic regression model adjusting for the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, a classification system to reflect the severity of disease upon admission to an ICU in the hospital (68), the miR-181b level was independently associated with sepsis (odds ratio [OR] 0.49, 95% CI 0.25–0.95, P = 0.03) and sepsis plus sepsis/ARDS (OR 0.52, 95% CI 0.29–0.92, P = 0.03). Thus, after adjusting for APACHE II score, each one unit increase in miR-181b level was associated with an approximately 50% decrease in the odds of having sepsis. The characteristics of control subjects and septic patients are shown in Table 2. Collectively, these data indicate that reduced circulating miR-181b levels in the plasma are associated with patients with sepsis.
Circulating miR-181b levels are reduced in plasma from patients with sepsis or sepsis/ARDS. (A) Circulating miR-181b in either patients with sepsis (n = 26) or control subjects admitted to the ICU without sepsis (n = 16). (B) Circulating miR-181b in patients with sepsis plus sepsis/ARDS (n = 36) compared with control subjects (n = 16). The expression levels of miR-181b were detected by real-time qPCR. Data show mean ± SEM. *P < 0.05.
The characteristics of control subjects and septic patients