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• W3124769289 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Endothelial cell (EC) activation is an early hallmark in the pathogenesis of chronic vascular diseases. MicroRNA-181b (Mir181b) is an important anti-inflammatory mediator in the vascular endothelium affecting endotoxemia, atherosclerosis, and insulin resistance. Herein, we identify that the drug methotrexate (MTX) and its downstream metabolite adenosine exert anti-inflammatory effects in the vascular endothelium by targeting and activating Mir181b expression. Both systemic and endothelial-specific Mir181a2b2-deficient mice develop vascular inflammation, white adipose tissue (WAT) inflammation, and insulin resistance in a diet-induced obesity model. Moreover, MTX attenuated diet-induced WAT inflammation, insulin resistance, and EC activation in a Mir181a2b2-dependent manner. Mechanistically, MTX attenuated cytokine-induced EC activation through a unique adenosine-adenosine receptor A3-SMAD3/4-Mir181b signaling cascade. These findings establish an essential role of endothelial Mir181b in controlling vascular inflammation and that restoring Mir181b in ECs by high-dose MTX or adenosine signaling may provide a potential therapeutic opportunity for anti-inflammatory therapy. Introduction Activated endothelial cells (ECs) orchestrate the expression of adhesion molecules and release chemokines to foster the recruitment of leukocytes into the vessel wall. This presents an early hallmark that contributes to the development of chronic vascular disease states, such as atherosclerosis, insulin resistance, and rheumatoid arthritis (RA) (Khan et al., 2010; Rao et al., 2007; Speyer and Ward, 2011; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016). Accumulating studies demonstrate that targeting EC activation and/or dysfunction may open new therapeutic approaches for the prevention and treatment of inflammatory diseases (Chen et al., 2017; Gareus et al., 2008; Nus et al., 2016). For example, inhibition of NF-κB activity specifically in the vascular endothelium significantly reduced atherosclerotic plaque formation and progression (Gareus et al., 2008). MicroRNAs (miRNAs), a class of evolutionary conserved small non-coding RNAs, are important post-transcriptional regulators of gene expression through mRNA degradation or translational repression (Loyer et al., 2014; Sun et al., 2013). We have previously identified microRNA-181b (Mir181b), an anti-inflammatory miRNA, which prevents EC activation and diverse vascular inflammatory disease states including endotoxemia, atherosclerosis, insulin resistance, and arterial thrombosis (Lin et al., 2016; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016), suggesting that Mir181b could serve as a promising target for preventing EC activation and dysfunction associated with inflammatory diseases. Mir181b expression is significantly reduced in response to a range of pro-inflammatory stimuli in the vascular endothelium (Lin et al., 2016; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016). Therefore, identification of signaling pathways or therapeutics to rescue Mir181b expression in the vascular endothelium could open new strategies to control vascular inflammation. Methotrexate (MTX) is a first-line treatment in RA and other autoimmune-mediated inflammatory diseases. Observational studies suggested that low-dose MTX could reduce cardiovascular risk in patients with RA (Charles-Schoeman et al., 2017; Deyab et al., 2017; Hjeltnes et al., 2013; Kisiel et al., 2015). For example, the Psoriatic arthritis, Ankylosing spondylitis, Rheumatoid Arthritis Study (PSARA) demonstrated that patients receiving MTX improved endothelial function and reduced E-selectin plasma levels after 6 months follow-up (Deyab et al., 2017; Hjeltnes et al., 2013). Another clinical trial identified that MTX treatment significantly lowered carotid intima-media thickness in RA patients compared to placebo (Kisiel et al., 2015). However, the recent Cardiovascular Inflammation Reduction Trial (CIRT), designed to determine the effect of low-dose MTX on cardiovascular secondary prevention, found that low-dose MTX did not reduce cardiovascular events compared to placebo (Ridker et al., 2019). These conflicting results from different clinical trials raise questions for the mechanisms of MTX-mediated effects on vascular inflammation. While adenosine has been identified as a potential metabolite activated in response to MTX in ECs (Haskó and Cronstein, 2013), gaps remain in our understanding of the downstream signaling pathways underlying its effects. In this study, we identify that high-dose MTX and its downstream metabolite adenosine exert anti-inflammatory effects in the vascular endothelium through an MTX-adenosine receptor A3-Mir181b-dependent signaling pathway. These results provide a general paradigm for therapeutic control of miRNA expression and that restoring Mir181b expression in the vascular endothelium by high-dose MTX or adenosine signaling may provide therapeutic opportunities for anti-inflammatory therapy. Results MTX attenuates TNF-α-induced EC activation by upregulation of MIR181B-2 expression Because of their known anti-inflammatory effects in the vascular endothelium (Diamantis et al., 2017; Mangoni et al., 2017), we explored whether MTX or HMG-CoA reductase inhibitors (statins) activated the expression of the anti-inflammatory MIR181B (Lin et al., 2016; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016). MTX (10 µM) but not statins significantly upregulated MIR181B expression by 1.35-fold in cultured human umbilical vein endothelial cells (HUVECs) (Figure 1A). The MTX-induced expression of MIR181B was dose- and time-dependent with the highest induction at 10 µM and 4 hr (Figure 1B,C). Previous studies have established that adenosine (Ad) is the major anti-inflammatory effector of MTX, which is a product of adenosine monophosphate, a reaction catalyzed by the enzyme ecto-5’ nucleotidase (Chan and Cronstein, 2013; Gadangi et al., 1996; Tian and Cronstein, 2007). When using a competitive enzyme inhibitor of ecto-5’ nucleotidase (α, β-methylene adenosine-5’-diphosphate (APCP)), the MTX-induced MIR181B expression was completely blocked (Figure 1D). Conversely, Ad alone increased MIR181B expression in a dose- and time-dependent manner with the highest observed increase by 2.3-fold at 50 µM and 4 hr (Figure 1E,F). Figure 1 with 1 supplement see all Download asset Open asset Methotrexate (MTX) represses TNF-α-induced pro-inflammatory gene expression via upregulation of MIR181B-2 expression in ECs. (A) Real-time qPCR analysis of mature MIR181B in HUVECs in the presence or absence of MTX (10 µM), Atorvastatin (1 µM), Mevastatin (1 µM), Simvastatin (1 µM), and Rosuvastatin (1 µM) for 4 hr. three biological replicates. Unpaired two-tailed Student t test. (B) Titration of MTX (0 to 100 µM) for 4 hr (three biological replicates, Unpaired two-tailed Student t test) and (C) time course of MTX (10 µM) in HUVECs to assess MIR181B expression, three biological replicates. One-way ANOVA. (D) Real-time qPCR analysis of MIR181B in HUVECs incubated with MTX (10 µM) or in combination with APCP (50 µM) for 4 hr. Three biological replicates. Unpaired two-tailed Student t test. (E) Dose-response of adenosine (Ad) (0 to 100 µM) for 4 hr and (F) time course of Ad (50 µM) over 0–12 hr on MIR181B expression in HUVECs. Three biological replicates. One-way ANOVA. (G) HUVECs were treated with TNF-α (10 ng/ml) alone or in combination with either MTX (10 µM) or Ad (50 µM) for 4 hr. Three biological replicates. Unpaired two-tailed Student t test. Analysis of primary transcript of (H) MIR181B-2 (Pri-MIR181B-2) or (I) MIR181A-2 (Pri-MIR181A-2) in response to TNF-α (10 ng/ml) with or without MTX (10 µM) or Ad (50 µM) for 4 hr in HUVECs. Three biological replicates. Unpaired two-tailed Student t test. (J) Isolated primary lung endothelial cells (ECs) from Mirr181a2b2flox/flox (flox) mice and Mir181a2b2-/- (KO) mice were treated with TNF-α (10 ng/ml) with or without MTX (10 µM) or Ad (50 µM) for 8 hr to analyze VCAM-1 protein expression. Please see Figure 1—source data 1. Three biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s. indicated non significance. All values represent mean ± SEM. Figure 1—source data 1 https://cdn.elifesciences.org/articles/58064/elife-58064-fig1-data1-v2.xlsx Download elife-58064-fig1-data1-v2.xlsx MIR181B expression is rapidly reduced in response to pro-inflammatory stimuli such as TNF-α in ECs and overexpression MIR181B suppressed TNF-α-induced EC activation by repressing VCAM-1, E-selectin, and ICAM-1 expression (MICU Registry et al., 2012; Yamasaki et al., 2003). To address whether MTX or Ad may rescue the TNF-α-mediated reduction in MIR181B expression, HUVECs were stimulated with TNF-α in combination with either MTX or Ad. Treatment with MTX or Ad fully rescued the TNF-α repression of MIR181B expression (Figure 1G). As expected, ECs treated with MTX or Ad significantly reduced VCAM-1, ICAM-1, and E-selectin expression stimulated by TNF-α (Figure 1—figure supplement 1A,B). Because two loci for MIR181B (i.e. MIR181B-1 and MIR181B-2) exist (Sun et al., 2014b), we examined which locus is regulated by TNF-α, MTX, or Ad. To this end, primary miRNA transcripts were analyzed in response to stimulation with TNF-α. Only primary transcript of the MIR181B-2 locus, and not primary MIR181B-1, MiR-181A-1, or MiR-181A-2, was rescued by MTX or Ad co-stimulation (Figure 1H,I; Figure 1—figure supplement 1C,D). To study the relationship between MTX or Ad to Mir181b in mouse tissues, primary lung ECs were isolated from Mir181a2b2—/— knockout mice (KO), reflecting deficiency of Mir181a-2 and Mir181b-2, or control Mir181a2b2flox/flox (flox). Because Mir181a-2 and Mir181b-2 are located in close proximity to each other, the knockout strategy deleted both miRNAs (Henao-Mejia et al., 2013). Isolated primary ECs were stimulated with TNF-α in the presence or absence of MTX or Ad. In ECs isolated from wild-type flox mice, MTX or Ad inhibited VCAM-1 protein expression by 40% and 35%, respectively. However, MTX or Ad did not reduce VCAM-1 expression in primary ECs from Mir181a2b2—/— KO mice (Figure 1J). Taken together, these findings suggest that pri-Mir181b-2 and its mature isoform Mir181b is increased by MTX or Ad, and may mediate the protective anti-inflammatory effects of MTX or Ad on TNF-α-induced EC activation. Adenosine receptor A3 (ADORA3) mediates MTX-induced expression of MIR181B in ECs Ad is released by cells to the extracellular environment at a low concertation and acts as a local modulator with a generally cytoprotective function through interaction with its four cell surface Ad receptors, ADORA1, ADORA2A, ADORA2B, and ADORA3. Each of these Ad receptors have a unique pharmacological profile, tissue distribution, and effector coupling (Haskó and Cronstein, 2004; Jacobson and Gao, 2006). To examine which Ad receptor mediates the MTX-induced MIR181B expression, individual knockdown for each of the Ad receptors was performed in ECs first in the absence of MTX or Ad (Figure 2—figure supplement 1A–H). Only silencing of ADORA3 reduced MIR181B basal expression by 40%, while knockdown of ADORA1, ADORA2A, and ADORA2B had no effect on MIR181B expression (Figure 2A). This suggests that ADORA3 might mediate the MIR181B expression activated by MTX or Ad. Therefore, we next examined whether AdoRA3 knockdown blocked the MTX or Ad-mediated increase in MIR181B expression. Indeed, neither MTX nor Ad increased MIR181B expression in ADORA3-deficient HUVECs compared to negative control siRNA (Figure 2B). MIR181B expression is reduced in response to pro-inflammatory stimuli such as TNF-α (Lin et al., 2016; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016). Treatment of ECs with TNF-α in the presence of MTX or Ad rescued the TNF-α-mediated repression of MIR181B expression in an AdoRA3-specific manner (Figure 2C and Figure 2—figure supplement 1A). Furthermore, silencing of ADORA3, (and not ADORA1, ADORA2A, or ADORA2B) abrogated the anti-inflammatory effect of MTX or Ad in TNF-α-activated ECs on VCAM-1, ICAM-1, and E-Selection expression (Figure 2D and Figure 2—figure supplement 1B). Finally, to further assess whether the anti-inflammatory effect of MTX or Ad is mediated through an ADORA3-MIR181B signaling cascade, knockdown of ADORA3 was combined with simultaneous overexpression of MIR181B mimic. While the MTX or Ad anti-inflammatory effects were blocked in the presence of ADORA3 silencing, overexpression of MIR181B fully rescued the MTX and Ad-mediated reduction of ECs activation markers (Figure 2E). In summary, our data indicate that ADOR3 plays central role in mediating MTX- or Ad-induced MIR181B expression, and this ADORA3-MIR181B signaling pathway contributes to the inhibitory effects of MTX or Ad on TNF-α-induced EC activation. Figure 2 with 1 supplement see all Download asset Open asset Induction of MIR181B expression by methotrexate (MTX) or adenosine is adenosine receptor A3 (ADORA3) dependent. (A) Knockdown for adenosine receptors A1, A2A, A2B, and A3 in HUVECs was performed to analyze MIR181B expression. three biological replicates. One-way ANOVA. (B) MIR181B expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA after treatment with MTX (10 µM) or Ad (50 µM) or (C) treatment with TNF-α (10 ng/ml) alone or in combination MTX (10 µM) or Ad (50 µM). Three biological replicates. One-way ANOVA and Unpaired two-tailed Student t test. (D) Western blot analyses of VCAM-1, ICAM-1, and E-Selectin expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA in the presence of TNF-α (10 ng/ml) in combination with either MTX (10 µM) or Ad (50 µM). Three biological replicates. Unpaired two-tailed Student t test. (E) in the presence of miRNA negative control (NS-m) or MIR181B mimics (181b-m) stimulated with TNF-α (10 ng/ml) or in combination with MTX (10 µM) or Ad (50 µM). Please see Figure 2—source data 1. Three biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s. indicated non significance. All values represent mean ± SEM. Figure 2—source data 1 https://cdn.elifesciences.org/articles/58064/elife-58064-fig2-data1-v2.xlsx Download elife-58064-fig2-data1-v2.xlsx MTX and Ad regulate MIR181B-2 promoter activity via SMAD3/4 To address, how MTX regulates MIR181B-2 expression on the transcriptional level, different lengths of the proximal MIR181A2B2 promoter (containing sequences 5’ to both MIR181A-2 and MIR181B-2) were cloned upstream of a luciferase reporter. MTX and Ad activated the −606 bp MIR181A2B2 promoter reporter construct from 1.47- to 1.57-fold, respectively (Figure 3A–B). Treatment of MTX or Ad similarly activated the −402 bp reporter comparable to the full-length constructs (Figure 3B). However, reducing the promoter length to −301 bp or −150 bp significantly reduced luciferase activity from 30- to 2600-fold, respectively (Figure 3B). At the post-transcriptional level, mRNA stability is an important factor determining mRNA abundance. To investigate whether MTX or Ad would affect pri-MIR181B-1 or pri- MIR181B-2 transcript stability, we used the transcription inhibitor actinomycin D in MTX or Ad-treated HUVECs. We found that neither MTX nor Ad affected the stability of pri-MIR181B-1 transcript (Figure 3—figure supplement 2A). However, MTX significantly increased pri-MIR181B-2 transcript stability, while Ad had a modest opposite effect (Figure 3—figure supplement 2B). These results indicate that the promoter region between −402 and −301 is likely required for MTX and Ad-induced transcriptional activity of the MIR181A2B2 locus and MTX and Ad affects pri-MIR181B-2 expression at both transcriptional and post-transcriptional levels. To further identify specific transcription factor binding sites, in silico prediction tools identified a SMAD2/3/4 binding site in the region −402 to −301 (Figure 3B). This was further investigated by individual knockdown of SMAD2, SMAD3, or SMAD4 (Figure 3—figure supplement 3). Silencing of SMAD3 or SMAD4 reduced luciferase activity of the full length −606 bp construct by 37% and 42%, respectively, in ECs compared to control siRNA (Figure 3C). However, SMAD2 silencing did not impact luciferase activity (Figure 3C). Consistent with this observation, MIR181B expression was significantly reduced only by silencing SMAD3 or SMAD4, and not by SMAD2 knockdown (Figure 3D). Importantly, silencing of SMAD3 or SMAD4 completely blocked the MTX and Ad-mediated increase in MIR181B expression (Figure 3E). Furthermore, knockdown ADORA3 blocked the MIR181A2B2 promoter luciferase activity induced by MTX and Ad (Figure 3F); however, silencing of the other three Ad receptors had no effect on luciferase activity (Figure 3G). Taken together, these data indicate that SMAD3 and SMAD4 are not only required for transcriptional activity of MIR181B expression, but also significantly contribute to MTX and Ad-induced MIR181B expression. Figure 3 with 3 supplements see all Download asset Open asset MIR181A2B2 promoter analysis in response to methotrexate (MTX) or Ad in endothelial cells (ECs). (A) Luciferase reporter constructs containing the indicated (0.606–5.1 kb 5’upstream) MIR181A2B2 promoter sequences were transfected in HEK 293 T cells and luciferase activity was measured 12 hr after treatment with MTX (10 µM) or Ad (50 µM), respectively. eight biological replicates. One-way ANOVA. (B) Luciferase reporter constructs containing the indicated (0.606 kb 5’upstream) MIR181A2B2 promoter sequences were transfected in HEK 293 T cells and luciferase activity was measured 12 hr after treatment with MTX (10 µM) or Ad (50 µM), respectively. Seven to eight biological replicates. One-way ANOVA. (C) Effect of siRNA-mediated knockdown for SMAD2, SMAD3, or SMAD4 in response to MTX or Ad on the 0.606 kb luciferase reporter. Three biological replicates. Unpaired two-tailed Student t test. (D–E) Real-time qPCR analysis of MIR181B expression in HUVECs transfected with siRNAs to negative control, SMAD2, SMAD3, or SMAD4 in the (D) absence or (E) presence of MTX (10 µM) or Ad (50 µM) for 4 hr. three biological replicates. Unpaired two-tailed Student t test. (F–G) Luciferase reporters containing the 0.606 kb miR-181a2b2 promoter were transfected in combination with siRNA to negative control or ADORA3 (F) or ADORA1, ADORA2A, or ADORA2B (G) and stimulated with MTX (10 µM) or Ad (50 µM). Please see Figure 3—source data 1. four biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. All values represent mean ± SEM. Figure 3—source data 1 https://cdn.elifesciences.org/articles/58064/elife-58064-fig3-data1-v2.xlsx Download elife-58064-fig3-data1-v2.xlsx MTX improves insulin sensitivity and epididymal white adipose tissue (eWAT) inflammation in diet-induced obese mice, but not in Mir181a2b2-/- mice Previous reports suggested that MTX can ameliorate diet-induced insulin resistance and inflammation (DeOliveira et al., 2012; Myers et al., 2017), although the mechanisms remained poorly understood. To evaluate the effective dose of MTX on Mir181b expression in vivo, C57Bl6 mice were injected with 0.5 mg/kg or 1.0 mg/kg of MTX. Low-dose (i.e. 0.25 mg/kg) MTX showed no significant increase in Mir181b expression in plasma, liver, and intima, but 1 mg/kg did (Figure 4—figure supplement 1A). Moreover, C57Bl6 mice treated with adenosine exhibited significantly elevated Mir181b expression in plasma by 168% and was even more pronounced than high-dose MTX (Figure 4—figure supplement 1B). In accordance with this dose-response of MTX on Mir181b expression, we examined whether the effect of MTX (1 mg/kg) on insulin resistance and adipose tissue inflammation is dependent on Mir181b2 expression in vivo. To this end Mir181a2b2flox/flox (flox) and Mir181a2b2—/— (KO) mice were placed on a 60% high-fat diet (HFD) for 12 weeks. Both flox and KO mice were injected with vehicle or MTX for 12 weeks (1 mg/kg/week by intraperitoneal injection), and insulin tolerance testing (ITT) and glucose tolerance testing (GTT) were performed (Figure 4A). As shown in Figure 4B, the body weights among the four groups were not significantly different, which suggest that the gain of body weight was independent of Mir181a2b2 expression and MTX treatment. However, MTX treatment significantly improved insulin sensitivity and reduced the area under curves (AUC) for ITT by 23% (Figure 4C) compared with vehicle injected flox mice, but not in glucose tolerance (Figure 4D). In line with our previous report showing a protective effect of Mir181b mimics on insulin resistance (Sun et al., 2016), KO mice developed insulin resistance (IR) as shown by improved insulin sensitivity (increased AUC for ITT by 121%) and glucose tolerance (increased AUC GTT by 122%) (Figure 4C,D). Notably, the beneficial effect of MTX on insulin sensitivity was completely blocked in KO mice (Figure 4C,D). Moreover, flox mice treated with MTX had increased insulin signaling activity in eWAT and liver compared vehicle injected, indicated by an increase in phosphorylation of Akt (phospho-Akt) in eWAT (by 23%) and in liver (by 34%) (Figure 4E). In contrast, KO mice without MTX injections showed a significant reduction of Akt phosphorylation by 24% in eWAT and 44% in liver compared with control flox mice (Figure 4E). Moreover, the MTX-mediated upregulation of phospho-Akt in eWAT and liver was blocked in KO mice (Figure 4E). However, MTX did not increase phospho-Akt levels in skeletal muscle (SM) (Figure 4E). Taken together, these findings from systemic miR-181a2b2 KO mice suggest that MTX increases Akt phosphorylation in eWAT and liver through miR-181a2b2 expression, which contributes to improved insulin sensitivity. Figure 4 with 2 supplements see all Download asset Open asset Systemic Mir181a2b2 KO blocks methotrexate (MTX)-mediated insulin sensitivity and visceral fat inflammation in diet-induced obesity. (A) Schema of experimental procedure for Mir181a2b2 flox/flox (flox) and Mir181a2b2—/— (KO) mice that were placed on a 60% high-fat diet (HFD) for 12 weeks. Each group of mice was weekly i.p. injected with vehicle control or MTX (1 mg/kg). (B) Body weights were measured weekly. Blood glucose levels were measured at week 11 for (C) insulin tolerance testing (ITT) and on week 12 for (D) glucose tolerance testing (GTT) with calculated area under the curves (AUC), respectively. (E) Western blot analysis of Akt and pSer473-Akt in epididymal white adipose tissue (eWAT), liver, and skeletal muscle with quantification across n = 3 independent experiments. (F) Real-time qPCR analysis of VCAM-1 expression in eWAT. (B–F), n = 9–10 mice per group, one-way ANOVA. (G) Paraffin sections of eWAT were stained with Mac2 and the positive areas were quantified, n = 5 mice per group, one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001. All values represent mean ± SEM. To better characterize the underlying mechanisms how MTX and Mir181a2b2 affects insulin resistance, we performed next-generation sequencing of RNA isolated from eWAT of these HFD-fed Mir181a2b2 flox and KO mice with or without MTX treatment. Gene set enrichment analysis (GSEA) suggested that the most statistically significant [false discovery rate (FDR) < 0.5] altered genes in eWAT from Mir181a2b2 KO mice were linked to cell adhesion (e.g. platelet-endothelium-leukocyte interactions, cell-matrix interactions, and cell junctions), chemotaxis, and immune response (Figure 4—figure supplement 2A-B). In eWAT from MTX-treated Mir181a2b2 flox and KO mice, GSEA found that cell cycle, inflammation, and chemotaxis pathways were more affected by MTX (Figure 4—figure supplement 2C-D). Macrophage accumulation plays a critical role in obesity-associated IR (Chawla et al., 2011; McNelis and Olefsky, 2014) and overexpression of Mir181b did not directly regulate cell-intrinsic monocyte/macrophage migration, proliferation, and activation (Sun et al., 2016). Moreover, we confirmed that MTX administration reduced VCAM-1 mRNA expression by 36% in eWAT in flox mice, but not MTX-injected KO mice (Figure 4F,G). In line with reduced VCAM-1 expression, we observed that macrophage content, as indicated by Mac2 staining, was significantly reduced by 80% in eWAT in MTX-injected flox mice (Figure 4H). In contrast, KO mice treated with MTX showed no inhibitory effect on VCAM-1 mRNA expression and macrophage content in eWAT (Figure 4F,G). Taken together, these results suggest that MTX administration leads to reduced EC activation and macrophage accumulation in white adipose tissue, largely dependent on Mir181a2b2 expression. MTX improves insulin sensitivity in obese mice, but not in EC-specific Mir181a2b2 KO mice It is well-established that some miRNAs may have a cell-type and/or tissue-specific expression and function which can impact the regulation of gene networks (Gao et al., 2011; Sood et al., 2006; Thomou et al., 2017; Zhou et al., 2013). Mir181b plays a dominant role in the vascular endothelium (Lin et al., 2016; Sun et al., 2014a; MICU Registry et al., 2012; Sun et al., 2016); however, its EC-specific genetic deletion has not been previously described. To assess whether endothelial Mir181a2b2 is mediating the observed phenotypes with respect to MTX and insulin sensitivity, we generated inducible EC-specific Mir181a2b2 mice (iEC-KO) by breeding the Mir181a2b2flox/flox mice to tamoxifen-inducible endothelial-specific Cre (vascular endothelial cadherin promoter [VECad-Cre-ERT2]) (Figure 5A). Compared to vehicle control injected mice, Mir181b expression in tamoxifen injected iEC-KO mice was reduced by 60% and 80% in aortic intima and ECs isolated from SM, respectively, but not in the aortic media and non-EC fraction of SM (Figure 5—figure supplement 1B–E). To assess whether endothelial Mir181a2b2 is driving the diet-induced IR and mediates MTX’s protective effects, we first examined MTX regulation of Mir181b expression. While Mir181b was reduced in the aortic intima of iEC-KO mice compared to flox-Cre, MTX increased Mir181b in flox-Cre, but not in iEC-KO mice (Figure 5—figure supplement 2A). Consistent with upregulated Mir181b expression, MTX reduced VCAM-1 expression in the intima of flox-Cre mice, but not in iEC-KO mice (Figure 5—figure supplement 2B). Because we identified that SMAD3/4 signaling contributed largely to Mir181b2 expression (Figure 3), we next investigated expression of SMADs in eWAT tissues from these HFD-fed flox-Cre and iEC-KO mice. We found that SMAD2 and SMAD3 expression decreased in MTX-treated iEC-KO mice compared to flox-Cre mice, whereas there were no differences between flox-Cre and iEC-KO mice without MTX treatment, suggesting a feedback mechanism involved in the MTX-SMAD-Mir181b cascade (Figure 5—figure supplement 3). Figure 5 with 3 supplements see all Download asset Open asset Endothelial cell (EC)-specific Mir181a2b2 KO blocks methotrexate (MTX)-mediated insulin sensitivity and visceral fat inflammation in diet-induced obesity. (A) Schema of experimental procedure for control Mir181a2b2flox/flox; VECad-Cre (flox-Cre) or tamoxifen injected EC-specific Mir181a2b2 KO (iEC-KO) mice that were placed on a 60% high-fat diet (HFD) for 15 weeks. Each group of mice was weekly i.p. injected with vehicle control or MTX (1 mg/kg). (B) Body weights over time of mice treated with vehicle or MTX, respectively. (C–D) ITT (C) and GTT (D) were measured and AUCs were quantified for each group. (E) Western blot analysis of Akt and pSer473-Akt in eWAT, liver, and skeletal muscle tissues. (F) Real-time qPCR analysis of VCAM-1 expression in eWAT. (G) Paraffin sections of eWAT were stained with Mac2 and the positive areas were quantified, n = 5 mice per group, one-way ANOVA. (B–G), n = 6 to 10 mice per group, one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001. All values represent mean ± SEM. As shown in Figure 5B, there were no significant differences in body weights between iEC-KO and control mice treated with or without MTX after 12 weeks of HFD. However, MTX administration significantly improved insulin sensitivity by 23% in ITT of control flox-Cre mice (Figure 5C), whereas MTX had no effect on insulin sensitivity in iEC-KO mice (Figure 5C). Similar to Mir181a2b2 systemic KO and control mice, MTX administration did not improve glucose tolerance either in iEC-KO or control mice (Figure 5D). Moreover, the MTX-mediated induction of Akt phosphorylation in eWATs and livers was blocked in iEC-KO mice (Figure 5E). Furthermore, MTX reduced VCAM-1 expression in eWAT in flox-Cre control mice, but not in iEC-KO mice (Figure 5F). Consistent with previous results (Figure 4H), MTX administration significantly reduced Mac2 positive macrophage content in eWAT tissues in flox-Cre mice, whereas no effect was observed in iEC-KO mice (Figure 5G). Finally, iEC-KO mice without MTX treatment showed a significant increase in Mac2 positive macrophages compared to flox-Cre control mice in eWAT tissues (Figure 5G). Taken together, these results identify a crucial role of endothelial Mir181a2b2 for mediating MTX’s anti-inflammatory effects. Discussion Low-grade inflammation in adipose tissue plays a critical role in the pathogenesis of obesity-associated insulin resistance, an important risk factor for the development of cardiovascular disease and type 2 diabetes (Arner and Kulyté, 2015). The Canakinumab Atherosclerosis Outcome Thrombosis Study (CANTOS) d" @default.
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• W3124769289 date "2020-12-29" @default.
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• W3124769289 title "Author response: Methotrexate attenuates vascular inflammation through an adenosine-microRNA-dependent pathway" @default.
• W3124769289 doi "https://doi.org/10.7554/elife.58064.sa2" @default.
• W3124769289 hasPublicationYear "2020" @default.
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